Methods of generating chimeric adenoviruses and uses for such chimeric aden oviruses

ABSTRACT

A method for providing an adenovirus from a serotype which does not grow efficiently in a desired cell line with the ability to grow in that cell line is described. The method involves replacing the left and right termini of the adenovirus with the corresponding termini from an adenovirus which grow efficiently in the desired cell line. At a minimum, the left terminus spans the (5′) inverted terminal repeat, the left terminus spans the E4 region and the (3′) inverted terminal repeat. The resulting chimeric adenovirus contains the internal regions spanning the genes encoding the penton, hexon and fiber from the serotype which does not grow efficiently in the desired cell. Also provided are vectors constructed from novel simian adenovirus sequences and proteins, host cells containing same, and uses thereof.

BACKGROUND OF THE INVENTION

The presence of humoral immunity (circulating antibodies) to adenoviruscapsid proteins is a barrier to the use of adenovirus vectors for genetherapy. The prototype adenovirus vectors that have been developed forgene therapy are based on subgroup C adenoviruses such as that ofserotype 5. The prevalence of neutralizing antibodies against subgroup Cadenoviruses is generally high in human populations as a result offrequent exposure to these pathogens. This fact is likely to greatlylimit the effectiveness of gene therapy vectors based on serotypes suchas Ad5.

Analysis of the nature of the protective antibodies against adenoviruseshas indicated that the most important target is the major capsidprotein, hexon [Wolfhart (1988) J. Virol 62, 2321; Gall et al. (1996) J.Virol. 70, 2116]. Several efforts have been made to engineer the hexonso as to evade the anti-hexon antibodies by making chimeric adenovirusesharboring hexons from other serotypes [Roy et al. (1998) J. Virol. 72,6875; U.S. Pat. No. 5,922,315; Gall et al. (1998) J. Virol. 72, 10260;Youil et al. (2002) Hum. Gene Ther. 13, 311; Wu et al. (2002) J. Virol.76, 12775]. However, this has been largely unsuccessful when exchangesamong distant serotypes are attempted.

Alternatively, investigators have proposed using adenovirus vectors thatrarely cause human infections or using adenoviruses from non-humansources. However, the lack of a practical manner in which to producelarge numbers of such vectors has proved to be a hindrance to developingsuch vectors.

SUMMARY OF THE INVENTION

The present invention provides a method of modifying adenoviruses havingcapsids, and particularly, including hexons, from serotypes which arenot well adapted for growth in cells useful for adenoviral virionproduction. The method is useful for production of scalable amounts ofadenoviruses. The resulting chimeric adenovirus genomes are useful for avariety of purposes which are described herein.

The invention further provides novel, isolated, adenovirus SA18 nucleicacid and amino acid sequences, vectors containing same, cell linescontaining such SA18 sequences and/or vectors, and uses thereof.

Other aspects and advantages of the present invention will be readilyapparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the map of the genome of the simian adenovirus generatedby shotgun cloning as described in the examples below.

FIG. 2 provides the map of the recombinant Adhu5-SV25 chimeric virus,termed H5S25H5.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides chimeric adenovirus genomes composed ofthe left terminal end and right terminal end of an adenovirus which canbe cultured in the selected host cell, and the internal regionsencoding, at a minimum, the capsid proteins of another adenovirusserotype. This invention is particularly advantageous for generatingadenoviruses having serotypes which are difficult to culture in adesired cell type. The invention thus permits generation of chimericadenoviruses vectors of varying serotypes.

In the embodiments illustrated herein, chimeric adenoviruses have beenconstructed where most structural proteins, and not merely the hexon orfiber, are derived from an adenovirus of an unrelated serotype, therebypreserving the majority of the protein-protein interactions that areinvolved in capsid assembly. Most of the early genes such as thoseencoded by the adenovirus E1 and E4 regions that are responsible fortranscription regulation and regulation of the host cell cycle, areretained from a different serotype that is known to result in high titervirus generation in the commonly used cell types, such as HEK 293 whichsupplies the Ad5 μl proteins in trans.

In another embodiment, the invention provides novel nucleic acid andamino acid sequences from Ad SA18, which was originally isolated fromvervet monkey [ATCC VR-943]. The present invention further providesnovel adenovirus vectors and packaging cell lines to produce thosevectors for use in the in vitro production of recombinant proteins orfragments or other reagents. The invention further provides compositionsfor use in delivering a heterologous molecule for therapeutic or vaccinepurposes. Such therapeutic or vaccine compositions contain theadenoviral vectors carrying an inserted heterologous molecule. Inaddition, novel sequences of the invention are useful in providing theessential helper functions required for production of recombinantadeno-associated viral (AAV) vectors. Thus, the invention provideshelper constructs, methods and cell lines which use these sequences insuch production methods.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or aprotein thereof, or a fragment thereof which is at least 8 amino acids,or more desirably, at least 15 amino acids in length. Examples ofsuitable fragments are described herein.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences thatare the same when aligned for maximum correspondence. The length ofsequence identity comparison may be over the full-length of the genome(e.g., about 36 kbp), the full-length of an open reading frame of agene, protein, subunit, or enzyme [see, e.g., the tables providing theadenoviral coding regions], or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments,e.g., of at least about nine nucleotides, usually at least about 20 to24 nucleotides, at least about 28 to 32 nucleotides, at least about 36or more nucleotides, may also be desired. Similarly, “percent sequenceidentity” may be readily determined for amino acid sequences, over thefull-length of a protein, or a fragment thereof. Suitably, a fragment isat least about 8 amino acids in length, and may be up to about 700 aminoacids. Examples of suitable fragments are described herein.

Identity is readily determined using such algorithms and computerprograms as are defined herein at default settings. Preferably, suchidentity is over the full length of the protein, enzyme, subunit, orover a fragment of at least about 8 amino acids in length. However,identity may be based upon shorter regions, where suited to the use towhich the identical gene product is being put.

As described herein, alignments are performed using any of a variety ofpublicly or commercially available Multiple Sequence Alignment Programs,such as “Clustal W”, accessible through Web Servers on the internet.Alternatively, Vector NTI utilities are also used. There are also anumber of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Similarly programs are available forperforming amino acid alignments. Generally, these programs are used atdefault settings, although one of skill in the art can alter thesesettings as needed. Alternatively, one of skill in the art can utilizeanother algorithm or computer program that provides at least the levelof identity or alignment as that provided by the referenced algorithmsand programs.

As used throughout this specification and the claims, the term“comprise” and its variants including, “comprises”, “comprising”, amongother variants, is inclusive of other components, elements, integers,steps and the like. The term “consists of” or “consisting of” areexclusive of other components, elements, integers, steps and the like.

Except where otherwise specified, the term “vector” includes any geneticelement known in the art which will deliver a target molecule to a cell,including, naked DNA, a plasmid, phage, transposon, cosmids, episomes,viruses, etc.

By “minigene” is meant the combination of a selected heterologous geneand the other regulatory elements necessary to drive translation,transcription and/or expression of the gene product in a host cell.

As used herein, the term “transcomplement” refers to when a gene (geneproduct) of one adenovirus serotype supplies an adenovirus serotypelacking this gene (gene product) from another serotype with the missingfunction. For example, human adenovirus serotype 5 E1a and E1b functionsare known to transcomplement E1-deleted chimpanzee adenovirus Pan 9.Similarly, the inventors have found that human Ad5 E1 transcomplementsE1-deleted chimpanzee adenovirus serotypes Pan5, Pan6, Pan7, and simianadenovirus serotypes SV1, SV25 and SV39. Other examples oftranscomplementing serotypes include human Ad5 and human Ad2, Ad3, Ad4,Ad5, Ad7, and Ad12.

The term “functionally deleted” or “functional deletion” means that asufficient amount of the gene region is removed or otherwise damaged,e.g., by mutation or modification, so that the gene region is no longercapable of producing functional products of gene expression. If desired,the entire gene region may be removed. Other suitable sites for genedisruption or deletion are discussed elsewhere in the application.

The term “functional” refers to a product (e.g., a protein or peptide)which performs its native function, although not necessarily at the samelevel as the native product. The term “functional” may also refer to agene which encodes and from which a desired product can be expressed.

I. Chimeric Adenoviral Vectors

The compositions of this invention include chimeric adenoviral vectorsthat deliver a heterologous molecule to cells. For delivery of such aheterologous molecule, the vector can be a plasmid or, preferably, achimeric adenovirus. The chimeric adenoviruses of the invention includeadenovirus DNA from at least two source serotypes, a “donating serotype”and a “parental adenovirus” as described in more detail herein, and aminigene.

Because the adenoviral genome contains open reading frames on bothstrands, in many instances reference is made herein to 5′ and 3′ ends ofthe various regions to avoid confusion between specific open readingframes and gene regions. Thus, when reference is made herein to the“left” and “right” end of the adenoviral genome, this reference is tothe ends of the approximately 36 kb adenoviral genome when depicted inschematic form as is conventional in the art [see, e.g., Horwitz,“Adenoviridae and Their Replication”, in VIROLOGY, 2d ed., pp. 1679-1721(1990)]. Thus, as used herein, the “left terminal end” of the adenoviralgenome refers to portion of the adenoviral genome which, when the genomeis depicted schematically in linear form, is located at the extreme leftend of the schematic. Typically, the left end refers to be portion ofthe genome beginning at map unit 0 and extending to the right to includeat least the 5′ inverted terminal repeats (ITRs), and excludes theinternal regions of the genome encoding the structural genes. As usedherein, the “right terminal end” of the adenoviral genome refers toportion of the adenoviral genome which, when the genome is depictedschematically in linear form, is located at the extreme right end of theschematic. Typically, the right end of the adenoviral genome refers tobe portion of the genome ending at map unit 36 and extending to the leftto include at least the 3′ ITRs, and excludes the internal regions ofthe genome encoding the structural genes.

A. Adenovirus Regulatory Sequences

1. Serotype

The selection of the adenovirus serotype donating its left terminal endand right terminal end can be readily made by one of skill in the artfrom among serotypes which can readily be cultured in the desired cellline. Among other factors which may be considered in selecting theserotype of the donating serotype is compatibility with the adenovirusserotype which will be supplying the internal regions at the location atwhich their sequences are hybridized.

Suitable adenoviruses for donating their left and right termini areavailable from the American Type Culture Collection, Manassas, Va., US(ATCC), a variety of academic and commercial sources, or the desiredregions of the donating adenoviruses may be synthesized using knowntechniques with reference to sequences published in the literature oravailable from databases (e.g., GenBank, etc.). Examples of suitabledonating adenoviruses include, without limitation, human adenovirusserotypes 2, 3, 4, 5, 7, and 12, and further including any of thepresently identified human types [see, e.g., Horwitz, “Adenoviridae andTheir Replication”, in VIROLOGY, 2d ed., pp. 1679-1721 (1990)] which canbe cultured in the desired cell. Similarly adenoviruses known to infectnon-human primates (e.g., chimpanzees, rhesus, macaque, and other simianspecies) or other non-human mammals and which grow in the desired cellcan be employed in the vector constructs of this invention. Suchserotypes include, without limitation, chimpanzee adenoviruses Pan 5[VR-591], Pan6 [VR-592], Pan7 [VR-593], and C68 (Pan9), described inU.S. Pat. No. 6,083,716; and simian adenoviruses including, withoutlimitation SV1 [VR-195]; SV25 [SV-201]; SV35; SV15; SV-34; SV-36; SV-37,and baboon adenovirus [VR-275], among others. The sequences of Pan 5(also termed C5), Pan 6 (also termed C6), Pan 7 (also termed C7), SV1,SV25, and SV39 have been described [WO 03/046124, published 5 Jun. 2003;and in U.S. patent application Ser. No. 10/739,096, filed Dec. 19,2003)], which are incorporated by reference. In the following examples,the human 293 cells and adenovirus type 5 (Ad5), Pan9, and Ad40 are usedfor convenience. However, one of skill in the art will understand thatother cell lines and/or comparable regions derived from other adenoviralstrains may be readily selected and used in the present invention in theplace of (or in combination with) these serotypes.

2. Sequences

The minimum sequences which must be supplied by the adenovirus donatingits left terminal end and its right terminal end include the 5′cis-elements and the 3′ cis-elements necessary for replication andpackaging. Typically, the 5′ cis-elements necessary for packaging andreplication include the 5′ inverted terminal repeat (ITR) sequences(which functions as origins of replication) and the native 5′ packagingenhancer domains (that contain sequences necessary for packaging linearAd genomes and enhancer elements for the E1 promoter). The right end ofthe adenoviral genome includes the 3′ cis-elements (including the ITRs)necessary for packaging and encapsidation. Desirably, the adenovirusserotype donating its left and right termini and/or an adenovirusserotype which transcomplements the serotype of the donating adenovirus,further provides the functions of the necessary adenovirus early genes,including E1 (E1a and E1b), E2 (E2a and E2b), and E4 (including at leastthe ORF6 region). E3 is not essential and may be deleted as desired,e.g., for insertion of a transgene in this region or to provide spacefor a transgene inserted in another region (typically for packaging itis desirable for the total adenoviral genome to be under 36 kb).

In certain embodiments, the necessary adenovirus early genes arecontained in the chimeric construct of the invention. In otherembodiment, one or more of the necessary adenovirus early genes can beprovided by the packaging host cell or in trans.

In general, the chimeric adenovirus of the invention contains regulatorysequences from the donating adenovirus serotype, or a transcomplementingserotype, to provide the chimeric adenovirus with compatible regulatoryproteins. Optionally, one or more of the necessary adenoviral structuralgenes is provided by the adenovirus donating its left terminal and itsright terminal end.

In certain embodiments, the chimeric adenovirus further contains one ormore functional adenovirus genes, including, the Endoprotease openreading frame, DNA binding protein, 100 kDa scaffolding protein, 33 kDaprotein, protein VIII, pTP, 52/55 kDa protein, protein VII, Mu and/orprotein VI from the adenovirus serotype donating its left and righttermini. Where all of these genes are derived from the adenovirusserotype donating the 5′ and 3′ ITRs, a “pseudotyped” virus is formed.In one embodiment, the chimeric adenovirus contains the left end of theadenovirus genome from the donating serotype, from the 5′ ITR throughthe end of the pol gene (or the pTP). In another embodiment, thechimeric adenovirus contains the left end of the donating adenovirusserotype, from the 5′ ITR through the penton. In yet another embodiment,the chimeric adenovirus contains the left end of the donating adenovirusserotype, e.g., through the end of pTP, but contains an ITR from anadenovirus serotype heterologous to the donating adenovirus serotype.Still other embodiments will be readily apparent from the presentdisclosure.

Optionally, one or more of the genes can be hybrids formed from thefusion of the donating adenovirus serotype and the parental adenovirusserotype providing the capsid proteins (e.g., without limitation,polymerase, terminal protein, IIIa protein). Suitably, these genesexpress functional proteins which permit packaging of the adenovirusgenes into the capsid. Alternatively, one or more of these proteins(whether hybrid or non-hybrid) can be functionally deleted in thechimeric adenovirus. Where desired, any necessary proteins functionallydeleted in the chimeric adenovirus can be expressed in trans in thepackaging cell.

B. Parental Adenovirus Structural Proteins

1. Serotypes

This invention is particularly well adapted for use in generatingchimeric adenoviruses in which the capsid proteins are from a parentaladenovirus which does not efficiently grow in a desirable host cell. Theselection of the parental adenovirus serotype providing the internalregions can be readily made by one of skill in the art based on theinformation provided herein.

A variety of suitable adenoviruses can serve as a parental adenovirussupplying the regions encoding the structural (i.e., capsid proteins).Many of such adenoviruses can be obtained from the same sources asdescribed above for the donating adenovirus serotypes. Examples ofsuitable parental adenovirus serotypes includes, without limitation,human adenovirus serotype 40, among others [see, e.g., Horwitz,“Adenoviridae and Their Replication”, in VIROLOGY, 2d ed., pp. 1679-1721(1990)], and adenoviruses known to infect non-human primates (e.g.,chimpanzees, rhesus, macaque, and other simian species) or othernon-human mammals, including, without limitation, chimpanzee adenovirusC1, described in U.S. Pat. No. 6,083,716, which is incorporated byreference; simian adenoviruses, and baboon adenoviruses, among others.In addition, the parental adenovirus supplying the internal regions maybe from a non-naturally occurring adenovirus serotype, such as may begenerated using a variety of techniques known to those of skill in theart.

In one embodiment illustrated herein, a chimeric virus that wasconstructed was that between the chimpanzee adenoviruses Pan-5 and C1exhibited a higher titer in human 293 cells than the wild-type parentalvirus. However, the invention is not limited to the use of thesechimpanzee adenoviruses, or to the combination of simian-simian,human-human, or simian-human chimeric adenoviruses. For example, it maybe desirable to utilize bovine or canine adenoviruses, or othernon-human mammalian adenoviruses which do not naturally infect and/orreplicate in human cells.

In the following examples, the human adenovirus type 40 (Ad40) and thechimpanzee adenovirus C1, simian Pan 5 and Ad40, and Pan 5 and simianadenovirus SA18, are used. However, one of skill in the art willunderstand that other adenoviral serotypes may be readily selected andused in the present invention in the place of (or in combination with)these serotypes.

2. Sequences

The parental adenovirus provides to the chimeric construct of theinvention its internal regions which includes structural proteinsnecessary for generating a capsid having the desired characteristics ofthe parental adenovirus. These desired characteristics include, but arenot limited to, the ability to infect target cells and delivery aheterologous transgene, the ability to elude neutralizing antibodiesdirected to another adenovirus serotype (i.e., avoiding clearance due tocross-reactivity), and/or the ability to infect cells in the absence ofan immune response to the chimeric adenovirus. The advantages of suchcharacteristics may be most readily apparent in a regimen which involvesrepeat delivery of adenoviral vectors. The left and right termini of theparent adenovirus, including at least the 5′ ITRs, the E1 region, the E4region and the 3′ ITRs are non-functional and, preferably, completelyabsent. Optionally, all adenovirus regulatory proteins from thisparental adenovirus are non-functional and only the structural proteins(or selected structural proteins) are retained.

At a minimum, the parental adenovirus provides the adenoviral lateregion encoding the hexon protein. Suitably, the parental adenovirusfurther provides the late regions encoding the penton and the fiber. Incertain embodiments, all of the functional adenoviral late regions,including L1 (encoding 52/55 Da, IIIa proteins), L2 (encoding penton,VII, V, Mu proteins), L3 (encoding VI, hexon, Endoprotease), L4(encoding 100 kD, 33 kD, VIII proteins) and L5 (encoding fiber protein)are supplied by the parental adenovirus. Optionally, one or more ofthese late gene functions, with the exception of those encoding thehexon, penton and fiber proteins, can be functionally deleted. Anynecessary structural proteins may be supplied in trans.

Thus, in certain embodiments, the chimeric adenovirus further containsone or more functional adenovirus genes, including, the Endoproteaseopen reading frame, DNA binding protein, 100 kDa scaffolding protein, 33kDa protein, protein VIII, pTP, 52/55 kDa protein, protein VII, Muand/or protein VI from the parental adenovirus donating its internalregions. Optionally, one or more of the genes can be hybrids formed fromthe fusion of the donating adenovirus serotype and the parentaladenovirus serotype providing the capsid proteins, as described above.

C. The “Minigene”

Typically, an adenoviral vector of the invention is designed to containa minigene which may be inserted into the site of a partially deleted,fully deleted (absent), or disrupted adenoviral gene. For example, theminigene may be located in the site of such a functional E1 deletion orfunctional E3 deletion, or another suitable site.

The methods employed for the selection of the transgene, the cloning andconstruction of the “minigene” and its insertion into the viral vectorare within the skill in the art given the teachings provided herein.

1. The transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the adenoviral vector will be put. For example, the adenoviralvector may be used as a helper virus in production of recombinantadeno-associated viruses or in production of recombinant adenovirusesdeleted of essential adenoviral gene functions which are supplied by theadenoviral vector, or for a variety of production uses. Alternatively,the adenoviral vector may be used for diagnostic purposes.

One type of transgene sequence includes a reporter sequence, which uponexpression produces a detectable signal. Such reporter sequencesinclude, without limitation, DNA sequences encoding β-lactamase,β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, greenfluorescent protein (GFP), chloramphenicol acetyltransferase (CAT),luciferase, membrane bound proteins including, for example, CD2, CD4,CD8, the influenza hemagglutinin protein, and others well known in theart, to which high affinity antibodies directed thereto exist or can beproduced by conventional means, and fusion proteins comprising amembrane bound protein appropriately fused to an antigen tag domainfrom, among others, hemagglutinin or Myc. These coding sequences, whenassociated with regulatory elements which drive their expression,provide signals detectable by conventional means, including enzymatic,radiographic, colorimetric, fluorescence or other spectrographic assays,fluorescent activating cell sorting assays and immunological assays,including enzyme linked immunosorbent assay (ELISA), radioimmunoassay(RIA) and immunohistochemistry. For example, where the marker sequenceis the LacZ gene, the presence of the vector carrying the signal isdetected by assays for beta-galactosidase activity. Where the transgeneis GFP or luciferase, the vector carrying the signal may be measuredvisually by color or light production in a luminometer.

However, desirably, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, or catalytic RNAs. Desirable RNA moleculesinclude tRNA, dsRNA, ribosomal RNA, si RNAs, small hairpin RNAs,trans-splicing RNAs, catalytic RNAs, and antisense RNAs. One example ofa useful RNA sequence is a sequence which extinguishes expression of atargeted nucleic acid sequence in the treated animal.

The transgene may be used for treatment, e.g., of genetic deficiencies,as a cancer therapeutic or vaccine, for induction of an immune response,and/or for prophylactic vaccine purposes. As used herein, induction ofan immune response refers to the ability of a molecule (e.g., a geneproduct) to induce a T cell and/or a humoral immune response to themolecule. The invention further includes using multiple transgenes,e.g., to correct or ameliorate a condition caused by a multi-subunitprotein. In certain situations, a different transgene may be used toencode each subunit of a protein, or to encode different peptides orproteins. This is desirable when the size of the DNA encoding theprotein subunit is large, e.g., for an immunoglobulin, theplatelet-derived growth factor, or a dystrophin protein. In order forthe cell to produce the multi-subunit protein, a cell is infected withthe recombinant virus containing each of the different subunits.Alternatively, different subunits of a protein may be encoded by thesame transgene. In this case, a single transgene includes the DNAencoding each of the subunits, with the DNA for each subunit separatedby an internal ribozyme entry site (IRES). This is desirable when thesize of the DNA encoding each of the subunits is small, e.g., the totalsize of the DNA encoding the subunits and the IRES is less than fivekilobases. As an alternative to an IRES, the DNA may be separated bysequences encoding a 2A peptide, which self-cleaves in apost-translational event. See, e.g., M. L. Donnelly, et al, J. Gen.

Virol., 78(Pt 1):13-21 (January 1997); Furler, S., et al, Gene Ther.,8(11):864-873 (June 2001); Klump H., et al., Gene Ther., 8(10):811-817(May 2001). This 2A peptide is significantly smaller than an IRES,making it well suited for use when space is a limiting factor. However,the selected transgene may encode any biologically active product orother product, e.g., a product desirable for study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis invention.

2. Vector and Transgene Regulatory Elements

In addition to the major elements identified above for the minigene, theadenoviral vector also includes conventional control elements which areoperably linked to the transgene in a manner that permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the invention.As used herein, “operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art. Forexample, inducible promoters include the zinc-inducible sheepmetallothionine (MT) promoter and the dexamethasone (Dex)-induciblemouse mammary tumor virus (MMTV) promoter. Other inducible systemsinclude the T7 polymerase promoter system [WO 98/10088]; the ecdysoneinsect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351(1996)], the tetracycline-repressible system [Gossen et al, Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system[Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al,Curr. Opin. Chem. Biol., 2:512-518 (1998)]. Other systems include theFK506 dimer, VP16 or p65 using castradiol, diphenol murislerone, theRU486-inducible system [Wang et al, Nat. Biotech., 15:239-243 (1997) andWang et al, Gene Ther., 4:432-441 (1997)] and the rapamycin-induciblesystem [Magari et al, J. Clin. Invest., 100:2865-2872 (1997)]. Theeffectiveness of some inducible promoters increases over time. In suchcases one can enhance the effectiveness of such systems by insertingmultiple repressors in tandem, e.g., TetR linked to a TetR by an IRES.Alternatively, one can wait at least 3 days before screening for thedesired function. One can enhance expression of desired proteins byknown means to enhance the effectiveness of this system. For example,using the Woodchuck Hepatitis Virus Posttranscriptional RegulatoryElement (WPRE).

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a transgene operably linkedto a tissue-specific promoter. For instance, if expression in skeletalmuscle is desired, a promoter active in muscle should be used. Theseinclude the promoters from genes encoding skeletal β-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase, as well as syntheticmuscle promoters with activities higher than naturally occurringpromoters (see L1 et al., Nat. Biotech., 17:241-245 (1999)). Examples ofpromoters that are tissue-specific are known for liver (albumin,Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus corepromoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), boneosteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bonesialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)),lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);immunoglobulin heavy chain; T cell receptor chain), neuronal such asneuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol.Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene (Piccioliet al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and theneuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)),among others.

Optionally, vectors carrying transgenes encoding therapeutically usefulor immunogenic products may also include selectable markers or reportergenes may include sequences encoding geneticin, hygromicin or purimycinresistance, among others. Such selectable reporters or marker genes(preferably located outside the viral genome to be packaged into a viralparticle) can be used to signal the presence of the plasmids inbacterial cells, such as ampicillin resistance. Other components of thevector may include an origin of replication. Selection of these andother promoters and vector elements are conventional and many suchsequences are available [see, e.g., Sambrook et al, and references citedtherein].

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts [Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.],use of overlapping oligonucleotide sequences of the adenovirus genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleotide sequence.

II. Production of the Recombinant Viral Particle

In one embodiment, the invention provides a method of generatingrecombinant chimeric adenoviral particles in which the capsid of thechimeric adenovirus is of a serotype incapable of efficient growth inthe selected host cell. A vector suitable for production of recombinantchimeric adenoviral particles can be generated by direct cloning.Alternatively, such particles can be generated by homologousrecombination between a first vector containing the left end of thechimeric adenoviral genome and a second vector containing the right endof the chimeric adenoviral genome. However, any suitable methodologyknown to those of skill in the art can be readily utilized to generate avector suitable to generate a production vector, preferably whichcontains the entire chimeric adenoviral genome, including a minigene.This production vector is then introduced into a host cell in which theadenoviral capsid protein is assembled and the chimeric adenoviralparticle assembled as described.

The chimeric adenoviruses of the invention include those in which one ormore adenoviral genes are absent, or otherwise rendered non-functional.If any of the missing gene functions are essential to the replicationand infectivity of the adenoviral particle, these functions are suppliedby a complementation (or transcomplementing) cell line or a helpervector expressing these functions during production of the chimericadenoviral particle.

Examples of chimeric adenoviruses containing such missing adenoviralgene functions include those which are partially or completely deletedin the E1a and/or E1b gene. In such a case, the E1 gene functions can besupplied by the packaging host cell, permitting the chimeric constructto be deleted of E1 gene functions and, if desired, for a transgene tobe inserted in this region. Optionally, the E1 gene can be of a serotypewhich transcomplements the serotype providing the other adenovirussequences in order to further reduce the possibility of recombinationand improve safety. In other embodiments, it is desirable to retain anintact E1a and/or E1b region in the recombinant adenoviruses. Such anintact E1 region may be located in its native location in the adenoviralgenome or placed in the site of a deletion in the native adenoviralgenome (e.g., in the E3 region).

In another example, all or a portion of the adenovirus delayed earlygene E3 may be eliminated from the chimeric adenovirus. The function ofadenovirus E3 is believed to be irrelevant to the function andproduction of the recombinant virus particle. Chimeric adenovirusvectors may also be constructed having a deletion of at least the ORF6region of the E4 gene, and more desirably because of the redundancy inthe function of this region, the entire E4 region. Still another vectorof this invention contains a deletion in the delayed early gene E2a.Similarly, deletions in the intermediate genes IX and IVa₂ may be usefulfor some purposes. Optionally, deletions may also be made in selectedportions of the late genes L1 through L5, as described above.

Other deletions may be made in the other structural or non-structuraladenovirus genes. The above-discussed deletions may be usedindividually, i.e., an adenovirus sequence for use in the presentinvention may contain deletions in only a single region. Alternatively,deletions of entire genes or portions thereof effective to destroy theirbiological activity may be used in any combination. For example, in oneexemplary vector, the adenovirus sequence may have deletions of the E1genes and the E4 gene, or of the E1, E2a and E3 genes, or of the E1 andE3 genes, or of E1, E2a and E4 genes, with or without deletion of E3,and so on. As discussed above, such deletions may be used in combinationwith other mutations, such as temperature-sensitive mutations, toachieve a desired result.

Examples of suitable transcomplementing serotypes are provided above.The use of transcomplementing serotypes can be particularly advantageouswhere there is diversity between the Ad sequences in the vector of theinvention and the human AdE1 sequences found in currently availablepackaging cells. In such cases, the use of the current humanE1-containing cells prevents the generation of replication-competentadenoviruses during the replication and production process. However, incertain circumstances, it will be desirable to utilize a cell line whichexpresses the E1 gene products can be utilized for production of anE1-deleted simian adenovirus. Such cell lines have been described. See,e.g., U.S. Pat. No. 6,083,716.

A. Packaging Host Cells

Suitably, the packaging host cell is selected from among cells in whichthe adenovirus serotype donating the left and right terminal ends of thechimeric genome are capable of efficient growth. The host cells arepreferably of mammalian origin, and most preferably are of non-humanprimate or human origin.

Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, cells such as A549 [ATCCAccession No. CCL 185], 911 cells, WEHI, 3T3, 10T1/2, HEK 293 cells orPERC6 (both of which express functional adenoviral E1) [Fallaux, F J etal, (1998), Hum Gene Ther, 9:1909-1917], Saos, C2C12, L cells, HT1080,HepG2, HeLa [ATCC Accession No. CCL 2], KB [CCL 17], Detroit [e.g.,Detroit 510, CCL 72] and WI-38 [CCL 75] cells, and primary fibroblast,hepatocyte and myoblast cells derived from mammals including human,monkey, mouse, rat, rabbit, and hamster. These cell lines are allavailable from the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209. Other suitable cell lines may beobtained from other sources. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

As described above, a chimeric adenovirus of the invention can lack oneor more functional adenoviral regulatory and/or structural genes whichare supplied either by the host cell or in trans to effect packaging ofthe chimeric adenovirus into the viral capsid to generate the viralparticle. Thus, the ability of a selected host cell to supplytranscomplementing adenoviral sequences may be taken into considerationin selecting a desired host cell.

In one example, the cells are from a stable cell line which expressesadenovirus E1a and E1b functions from a cell line which transcomplementsthe adenovirus serotype which donates the left and right termini to thechimera of the invention, permitting the chimera to be E1-deleted.Alternatively, where the cell line does not transcomplement theadenovirus donating the termini, E1 functions may be provided by thechimera, or in trans.

If desired, one may utilize the sequences provided herein to generate apackaging cell or cell line that expresses, at a minimum, the adenovirusE1 gene from the adenovirus serotype donating the 5′ ITR under thetranscriptional control of a promoter for expression, or atranscomplementing serotype, in a selected parent cell line. Inducibleor constitutive promoters may be employed for this purpose. Examples ofsuch promoters are described in detail elsewhere in this specification.A parent cell is selected for the generation of a novel cell lineexpressing any desired adenovirus or adenovirus gene, including, e.g., ahuman Ad5, AdPan5, Pan6, Pan7, SV1, SV25 or SV39 gene. Withoutlimitation, such a parent cell line may be HeLa [ATCC Accession No. CCL2), A549 [ATCC Accession No. CCL 185], HEK 293, KB [CCL 17], Detroit[e.g., Detroit 510, CCL 72] and WI-38 [CCL 75] cells, among others. Manyof these cell lines are all available from the ATCC. Other suitableparent cell lines may be obtained from other sources.

Such E1-expressing cell lines are useful in the generation of chimericadenovirus E1 deleted vectors. Additionally, or alternatively, theinvention provides cell lines that express one or more simian adenoviralgene products, e.g., E1a, E1b, E2a, and/or E4 ORF6, can be constructedusing essentially the same procedures for use in the generation ofchimeric viral vectors. Such cell lines can be utilized totranscomplement adenovirus vectors deleted in the essential genes thatencode those products, or to provide helper functions necessary forpackaging of a helper-dependent virus (e.g., adeno-associated virus).The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., cited above, use of overlapping oligonucleotidesequences of the adenovirus genomes, combined with polymerase chainreaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

In still another alternative, the essential adenoviral gene products areprovided in trans by the adenoviral vector and/or helper virus. In suchan instance, a suitable host cell can be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells, and eukaryoticcells, including, insect cells, yeast cells and mammalian cells.Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, cells such as A549, WEHI, 3T3, 1OT1/2, HEK 293 cells or PERC6 (both of which express functionaladenoviral E1) [Fallaux, F J et al, (1998), Hum Gene Ther, 9:1909-1917],Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

B. Helper Vectors

Thus, depending upon the adenovirus gene content of the adenoviralvectors and any adenoviral gene functions expressed from the host cell,a helper vector may be necessary to provide sufficient adenovirus genesequences necessary to produce an infective recombinant viral particlecontaining the minigene. See, for example, the techniques described forpreparation of a “minimal” human Ad vector in International PatentApplication WO96/13597, published May 9, 1996, and incorporated hereinby reference. Suitably, these helper vectors may be non-replicatinggenetic elements, a plasmid, or a virus.

Useful helper vectors contain selected adenovirus gene sequences notpresent in the adenovirus vector construct and/or not expressed by thepackaging cell line in which the vector is transfected. In oneembodiment, the helper virus is replication-defective and contains avariety of adenovirus genes in addition to the sequences describedabove. Such a helper vector is desirably used in combination with anE1-expressing cell line.

Helper vectors may be formed into poly-cation conjugates as described inWu et al, J. Biol. Chem., 264:16985-16987 (1989); K. J. Fisher and J. M.Wilson, Biochem. J., 299:49 (Apr. 1, 1994). A helper vector mayoptionally contain a second reporter minigene. A number of such reportergenes are known to the art. The presence of a reporter gene on thehelper virus which is different from the transgene on the adenovirusvector allows both the Ad vector and the helper vector to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

C. Assembly of Viral Particle and Transfection of a Cell Line

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, and preferably about 10 to about 50 μg DNA to about1×10⁴ cells to about 1×10¹³ cells, and preferably about 10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected.

Introduction into the host cell of the vector may be achieved by anymeans known in the art or as disclosed above, including transfection,and infection. One or more of the adenoviral genes may be stablyintegrated into the genome of the host cell, stably expressed asepisomes, or expressed transiently. The gene products may all beexpressed transiently, on an episome or stably integrated, or some ofthe gene products may be expressed stably while others are expressedtransiently.

Furthermore, the promoters for each of the adenoviral genes may beselected independently from a constitutive promoter, an induciblepromoter or a native adenoviral promoter. The promoters may be regulatedby a specific physiological state of the organism or cell (i.e., by thedifferentiation state or in replicating or quiescent cells) or byexogenously added factors, for example.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation.

Assembly of the selected DNA sequences of the adenovirus (as well as thetransgene and other vector elements) into various intermediate plasmids,and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques. Such techniquesinclude direct cloning as described [G. Gao et al, Gene Ther. 2003October; 10(22):1926-1930; US Patent Publication No. 2003-0092161-A,published May 15, 2003; International Patent Application No.PCT/US03/12405]. Other cloning techniques of cDNA such as thosedescribed in texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, and any suitable method which provides the desired nucleotidesequence can be utilized. Standard transfection and co-transfectiontechniques are employed, e.g., CaPO₄ precipitation techniques. Otherconventional methods employed include homologous recombination of theviral genomes, plaquing of viruses in agar overlay, methods of measuringsignal generation, and the like.

For example, following the construction and assembly of the desiredminigene-containing viral vector, the vector is transfected in vitro inthe presence of an optional helper vector into the packaging cell line.The functions expressed from the plasmid, packaging cell line and helpervirus, if any, permits the adenovirus-transgene sequences in the vectorto be replicated and packaged into virion capsids, resulting in thechimeric viral particles. The current method for producing such virusparticles is transfection-based. However, the invention is not limitedto such methods. The resulting chimeric adenoviruses are useful intransferring a selected transgene to a selected cell.

III. Use of the Chimeric Adenovirus Vectors

The chimeric adenovirus vectors of the invention are useful for genetransfer to a human or veterinary subject (including, non-humanprimates, non-simian primates, and other mammals) in vitro, ex vivo, andin vivo.

The recombinant adenovirus vectors described herein can be used asexpression vectors for the production of the products encoded by theheterologous genes in vitro. For example, the recombinant adenovirusescontaining a gene inserted into the location of an E1 deletion may betransfected into an E1-expressing cell line as described above.Alternatively, replication-competent adenoviruses may be used in anotherselected cell line. The transfected cells are then cultured in theconventional manner, allowing the recombinant adenovirus to express thegene product from the promoter. The gene product may then be recoveredfrom the culture medium by known conventional methods of proteinisolation and recovery from culture.

A chimeric adenoviral vector of the invention provides an efficient genetransfer vehicle that can deliver a selected transgene to a selectedhost cell in vivo or ex vivo even where the organism has neutralizingantibodies to one or more AAV serotypes. In one embodiment, the rAd andthe cells are mixed ex vivo; the infected cells are cultured usingconventional methodologies; and the transduced cells are re-infused intothe patient. These compositions are particularly well suited to genedelivery for therapeutic purposes and for immunization, includinginducing protective immunity.

More commonly, the chimeric adenoviral vectors of the invention will beutilized for delivery of therapeutic or immunogenic molecules, asdescribed below. It will be readily understood for both applicationsthat the recombinant adenoviral vectors of the invention areparticularly well suited for use in regimens involving repeat deliveryof recombinant adenoviral vectors. Such regimens typically involvedelivery of a series of viral vectors in which the viral capsids arealternated. The viral capsids may be changed for each subsequentadministration, or after a pre-selected number of administrations of aparticular serotype capsid (e.g., one, two, three, four or more). Thus,a regimen may involve delivery of a rAd with a first capsid, deliverywith a rAd with a second capsid, and delivery with a third capsid. Avariety of other regimens which use the Ad capsids of the inventionalone, in combination with one another, or in combination with other Adserotypes will be apparent to those of skill in the art. Optionally,such a regimen may involve administration of rAd with capsids ofnon-human primate adenoviruses, human adenoviruses, or artificial (e.g.,chimeric) serotypes such as are described herein. Each phase of theregimen may involve administration of a series of injections (or otherdelivery routes) with a single Ad serotype capsid followed by a serieswith another Ad serotype capsid. Alternatively, the recombinant Advectors of the invention may be utilized in regimens involving othernon-adenoviral-mediated delivery systems, including other viral systems,non-viral delivery systems, protein, peptides, and other biologicallyactive molecules.

The following sections will focus on exemplary molecules which may bedelivered via the adenoviral vectors of the invention.

A. Ad-Mediated Delivery of Therapeutic Molecules

In one embodiment, the Ad vectors described herein are administered tohumans according to published methods for gene therapy. A viral vectorof the invention bearing the selected transgene may be administered to apatient, preferably suspended in a biologically compatible solution orpharmaceutically acceptable delivery vehicle. A suitable vehicleincludes sterile saline. Other aqueous and non-aqueous isotonic sterileinjection solutions and aqueous and non-aqueous sterile suspensionsknown to be pharmaceutically acceptable carriers and well known to thoseof skill in the art may be employed for this purpose.

The adenoviral vectors are administered in sufficient amounts totransduce the target cells and to provide sufficient levels of genetransfer and expression to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the retina and other intraoculardelivery methods, direct delivery to the liver, inhalation, intranasal,intravenous, intramuscular, intratracheal, subcutaneous, intradermal,rectal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe transgene or the condition. The route of administration primarilywill depend on the nature of the condition being treated.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectiveadult human or veterinary dosage of the viral vector is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² particles. Dosageswill range depending upon the size of the animal and the route ofadministration. For example, a suitable human or veterinary dosage (forabout an 80 kg animal) for intramuscular injection is in the range ofabout 1×10⁹ to about 5×10¹² particles per mL, for a single site.Optionally, multiple sites of administration may be delivered. Inanother example, a suitable human or veterinary dosage may be in therange of about 1×10¹¹ to about 1×10¹⁵ particles for an oral formulation.One of skill in the art may adjust these doses, depending the route ofadministration, and the therapeutic or vaccinal application for whichthe recombinant vector is employed. The levels of expression of thetransgene, or for an immunogen, the level of circulating antibody, canbe monitored to determine the frequency of dosage administration. Yetother methods for determining the timing of frequency of administrationwill be readily apparent to one of skill in the art.

An optional method step involves the co-administration to the patient,either concurrently with, or before or after administration of the viralvector, of a suitable amount of a short acting immune modulator. Theselected immune modulator is defined herein as an agent capable ofinhibiting the formation of neutralizing antibodies directed against therecombinant vector of this invention or capable of inhibiting cytolyticT lymphocyte (CTL) elimination of the vector. The immune modulator mayinterfere with the interactions between the T helper subsets (T_(H1) orT_(H2)) and B cells to inhibit neutralizing antibody formation.Alternatively, the immune modulator may inhibit the interaction betweenT_(H1) cells and CTLs to reduce the occurrence of CTL elimination of thevector. A variety of useful immune modulators and dosages for use ofsame are disclosed, for example, in Yang et al., J. Virol., 70(9) (Sept1996); International Patent Application No. WO96/12406, published May 2,1996; and International Patent Application No. PCT/US96/03035, allincorporated herein by reference. Typically, such immune modulatorswould be selected when the transgene is a therapeutic which requiresrepeat delivery.

1. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), transforming growth factor α (TGF α), platelet-derived growthfactor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), anyone of the transforming growth factor superfamily, including TGF,activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs1-15, any one of the heregluin/neuregulin/ARIA/neu differentiationfactor (NDF) family of growth factors, nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4/5,ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophicfactor (GDNF), neurturin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors and, interferons,and, stem cell factor, flk-2/flt3 ligand. Gene products produced by theimmune system are also useful in the invention. These include, withoutlimitation, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimericimmunoglobulins, humanized antibodies, single chain antibodies, T cellreceptors, chimeric T cell receptors, single chain T cell receptors,class I and class II MHC molecules, as well as engineeredimmunoglobulins and MHC molecules. Useful gene products also includecomplement regulatory proteins such as complement regulatory proteins,membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1,CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation, including the low density lipoprotein (LDL)receptor, high density lipoprotein (HDL) receptor, the very low densitylipoprotein (VLDL) receptor, proteins useful in the regulation oflipids, including, e.g., apolipoprotein (apo) A and its isoforms (e.g.,ApoAI), apoE and its isoforms including E2, E3 and E4), SRB1, ABC1, andthe scavenger receptor. The invention also encompasses gene productssuch as members of the steroid hormone receptor superfamily includingglucocorticoid receptors and estrogen receptors, Vitamin D receptors andother nuclear receptors. In addition, useful gene products includetranscription factors such as jun, fos, max, mad, serum response factor(SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SPI,CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilmstumor protein, ETS-binding protein, STAT, GATA-box binding proteins,e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence. Other useful gene products include thoseuseful for treatment of hemophilia A (e.g., Factor VIII and itsvariants, including the light chain and heavy chain of the heterodimer,optionally operably linked by a junction), and the B-domain deletedFactor VIII, see U.S. Pat. Nos. 6,200,560 and 6,221,349], and useful fortreatment of hemophilia B (e.g, Factor IX).

Still other useful gene products include non-naturally occurringpolypeptides, such as chimeric or hybrid polypeptides having anon-naturally occurring amino acid sequence containing insertions,deletions or amino acid substitutions. For example, single-chainengineered immunoglobulins could be useful in certain immunocompromisedpatients. Other types of non-naturally occurring gene sequences includeantisense molecules and catalytic nucleic acids, such as ribozymes,which could be used to reduce overexpression of a target.

Reduction and/or modulation of expression of a gene are particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce self-directed antibodies. T-cellmediated autoimmune diseases include rheumatoid arthritis (RA), multiplesclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (DDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Each of these diseases is characterized by T cellreceptors (TCRs) that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases.

The chimeric adenoviral vectors of the invention are particularly wellsuited for therapeutic regimens in which multiple adenoviral-mediateddeliveries of transgenes is desired, e.g., in regimens involvingredelivery of the same transgene or in combination regimens involvingdelivery of other transgenes. Such regimens may involve administrationof a chimeric adenoviral vector, followed by re-administration with avector from the same serotype adenovirus. Particularly desirableregimens involve administration of a chimeric adenoviral vector of theinvention, in which the serotype of the viral vector delivered in thefirst administration differs from the serotype of the viral vectorutilized in one or more of the subsequent administrations. For example,a therapeutic regimen involves administration of a chimeric vector andrepeat administration with one or more adenoviral vectors of the same ordifferent serotypes. In another example, a therapeutic regimen involvesadministration of an adenoviral vector followed by repeat administrationwith a chimeric vector of the invention which differs from the serotypeof the first delivered adenoviral vector, and optionally furtheradministration with another vector which is the same or, preferably,differs from the serotype of the vector in the prior administrationsteps. These regimens are not limited to delivery of adenoviral vectorsconstructed using the chimeric serotypes of the invention. Rather, theseregimens can readily utilize chimeric or non-chimeric vectors of otheradenoviral serotypes, which may be of artificial, human or non-humanprimate, or other mammalian sources, in combination with one or more ofthe chimeric vectors of the invention. Examples of such serotypes arediscussed elsewhere in this document. Further, these therapeuticregimens may involve either simultaneous or sequential delivery ofchimeric adenoviral vectors of the invention in combination withnon-adenoviral vectors, non-viral vectors, and/or a variety of othertherapeutically useful compounds or molecules. The present invention isnot limited to these therapeutic regimens, a variety of which will bereadily apparent to one of skill in the art.

B. Ad-Mediated Delivery of Immunogenic Transgenes

The adenoviruses of the invention may also be employed as immunogeniccompositions. As used herein, an immunogenic composition is acomposition to which a humoral (e.g., antibody) or cellular (e.g., acytotoxic T cell) response is mounted to a transgene product deliveredby the immunogenic composition following delivery to a mammal, andpreferably a primate. The present invention provides an Ad that cancontain in any of its adenovirus sequence deletions a gene encoding adesired immunogen. Chimeric adenoviruses based on simian or othernon-human mammalian primate serotypes are likely to be better suited foruse as a live recombinant virus vaccine in different animal speciescompared to an adenovirus of human origin, but is not limited to such ause. The recombinant adenoviruses can be used as prophylactic ortherapeutic vaccines against any pathogen for which the antigen(s)crucial for induction of an immune response and able to limit the spreadof the pathogen has been identified and for which the cDNA is available.

Such vaccinal (or other immunogenic) compositions are formulated in asuitable delivery vehicle, as described above. Generally, doses for theimmunogenic compositions are in the range defined above for therapeuticcompositions. The levels of immunity of the selected gene can bemonitored to determine the need, if any, for boosters. Following anassessment of antibody titers in the serum, optional boosterimmunizations may be desired.

Optionally, a vaccinal composition of the invention may be formulated tocontain other components, including, e.g. adjuvants, stabilizers, pHadjusters, preservatives and the like. Such components are well known tothose of skill in the vaccine art. Examples of suitable adjuvantsinclude, without limitation, liposomes, alum, monophosphoryl lipid A,and any biologically active factor, such as cytokine, an interleukin, achemokine, a ligands, and optimally combinations thereof. Certain ofthese biologically active factors can be expressed in vivo, e.g., via aplasmid or viral vector. For example, such an adjuvant can beadministered with a priming DNA vaccine encoding an antigen to enhancethe antigen-specific immune response compared with the immune responsegenerated upon priming with a DNA vaccine encoding the antigen only.

The adenoviruses are administered in “an immunogenic amount”, that is,an amount of adenovirus that is effective in a route of administrationto transfect the desired cells and provide sufficient levels ofexpression of the selected gene to induce an immune response. Whereprotective immunity is provided, the recombinant adenoviruses areconsidered to be vaccine compositions useful in preventing infectionand/or recurrent disease.

Alternatively, or in addition, the vectors of the invention may containa transgene encoding a peptide, polypeptide or protein which induces animmune response to a selected immunogen. The recombinant adenoviruses ofthis invention are expected to be highly efficacious at inducingcytolytic T cells and antibodies to the inserted heterologous antigenicprotein expressed by the vector.

For example, immunogens may be selected from a variety of viralfamilies. Example of desirable viral families against which an immuneresponse would be desirable include, the picornavirus family, whichincludes the genera rhinoviruses, which are responsible for about 50% ofcases of the common cold; the genera enteroviruses, which includepolioviruses, coxsackieviruses, echoviruses, and human enterovirusessuch as hepatitis A virus; and the genera apthoviruses, which areresponsible for foot and mouth diseases, primarily in non-human animals.Within the picornavirus family of viruses, target antigens include theVP1, VP2, VP3, VP4, and VPG. Another viral family includes thecalcivirus family, which encompasses the Norwalk group of viruses, whichare an important causative agent of epidemic gastroenteritis. Stillanother viral family desirable for use in targeting antigens forinducing immune responses in humans and non-human animals is thetogavirus family, which includes the genera alphavirus, which includeSindbis viruses, RossRiver virus, and Venezuelan, Eastern & WesternEquine encephalitis, and rubivirus, including Rubella virus. Theflaviviridae family includes dengue, yellow fever, Japaneseencephalitis, St. Louis encephalitis and tick borne encephalitisviruses. Other target antigens may be generated from the Hepatitis C orthe coronavirus family, which includes a number of non-human virusessuch as infectious bronchitis virus (poultry), porcine transmissiblegastroenteric virus (pig), porcine hemagglutinatin encephalomyelitisvirus (pig), feline infectious peritonitis virus (cats), feline entericcoronavirus (cat), canine coronavirus (dog), and human respiratorycoronaviruses, which may cause the common cold and/or non-A, B or Chepatitis. In addition, the human coronaviruses include the putativecausative agent of sudden acute respiratory syndrome (SARS). Within thecoronavirus family, target antigens include the E1 (also called M ormatrix protein), E2 (also called S or Spike protein), E3 (also called HEor hemagglutin-elterose) glycoprotein (not present in allcoronaviruses), or N (nucleocapsid). Still other antigens may betargeted against the rhabdovirus family, which includes the generavesiculovirus (e.g., Vesicular Stomatitis Virus), and the generallyssavirus (e.g., rabies). Within the rhabdovirus family, suitableantigens may be derived from the G protein or the N protein. The familyfiloviridae, which includes hemorrhagic fever viruses such as Marburgand Ebola virus, may be a suitable source of antigens. The paramyxovirusfamily includes parainfluenza Virus Type 1, parainfluenza Virus Type 3,bovine parainfluenza Virus Type 3, rubulavirus (mumps virus),parainfluenza Virus Type 2, parainfluenza virus Type 4, Newcastledisease virus (chickens), rinderpest, morbillivirus, which includesmeasles and canine distemper, and pneumovirus, which includesrespiratory syncytial virus. The influenza virus is classified withinthe family orthomyxovirus and is a suitable source of antigen (e.g., theHA protein, the N1 protein). The bunyavirus family includes the generabunyavirus (California encephalitis, La Crosse), phlebovirus (RiftValley Fever), hantavirus (puremala is a hemahagin fever virus),nairovirus (Nairobi sheep disease) and various unassigned bungaviruses.The arenavirus family provides a source of antigens against LCM andLassa fever virus. The reovirus family includes the genera reovirus,rotavirus (which causes acute gastroenteritis in children), orbiviruses,and cultivirus (Colorado Tick fever), Lebombo (humans), equineencephalosis, blue tongue.

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp41, or smaller fragments thereof,e.g., of at least about 8 amino acids in length. Similarly, fragments ofthe tat protein may be selected. [See, U.S. Pat. No. 5,891,994 and U.S.Pat. No. 6,193,981.] See, also, the HIV and SIV proteins described in D.H. Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R. R.Amara, et al, Science, 292:69-74 (6 Apr. 2001). In another example, theHIV and/or SIV immunogenic proteins or peptides may be used to formfusion proteins or other immunogenic molecules. See, e.g., the HIV-1 Tatand/or Nef fusion proteins and immunization regimens described in WO01/54719, published Aug. 2, 2001, and WO 99/16884, published Apr. 8,1999. The invention is not limited to the HIV and/or SIV immunogenicproteins or peptides described herein. In addition, a variety ofmodifications to these proteins have been described or could readily bemade by one of skill in the art. See, e.g., the modified gag proteinthat is described in U.S. Pat. No. 5,972,596. Further, any desired HIVand/or SIV immunogens may be delivered alone or in combination. Suchcombinations may include expression from a single vector or frommultiple vectors. Optionally, another combination may involve deliveryof one or more expressed immunogens with delivery of one or more of theimmunogens in protein form. Such combinations are discussed in moredetail below.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/orenteritis. The parvovirus includes family feline parvovirus (felineenteritis), feline panleucopeniavirus, canine parvovirus, and porcineparvovirus. The herpesvirus family includes the sub-familyalphaherpesvirinae, which encompasses the genera simplexvirus (HSVI,HSVII), varicellovirus (pseudorabies, varicella zoster) and thesub-family betaherpesvirinae, which includes the genera cytomegalovirus(HCMV, muromegalovirus) and the sub-family gammaherpesvirinae, whichincludes the genera lymphocryptovirus, EBV (Burkitts lymphoma),infectious rhinotracheitis, Marek's disease virus, and rhadinovirus. Thepoxvirus family includes the sub-family chordopoxyirinae, whichencompasses the genera orthopoxvirus (Variola (Smallpox) and Vaccinia(Cowpox)), parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus,suipoxvirus, and the sub-family entomopoxyirinae. The hepadnavirusfamily includes the Hepatitis B virus. One unclassified virus which maybe suitable source of antigens is the Hepatitis delta virus. Still otherviral sources may include avian infectious bursal disease virus andporcine respiratory and reproductive syndrome virus. The alphavirusfamily includes equine arteritis virus and various Encephalitis viruses.

The viruses of the present invention may also carry immunogens which areuseful to immunize a human or non-human animal against other pathogensincluding bacteria, fungi, parasitic microorganisms or multicellularparasites which infect human and non-human vertebrates, or from a cancercell or tumor cell. Examples of bacterial pathogens include pathogenicgram-positive cocci include pneumococci; staphylococci; andstreptococci. Pathogenic gram-negative cocci include meningococcus;gonococcus. Pathogenic enteric gram-negative bacilli includeenterobacteriaceae; pseudomonas, acinetobacteria and eikenella;melioidosis; salmonella; shigella; haemophilus; moraxella; H. ducreyi(which causes chancroid); brucella; Franisella tularensis (which causestularemia); yersinia (pasteurella); streptobacillus moniliformis andspirillum; Gram-positive bacilli include listeria monocytogenes;erysipelothrix rhusiopathiae; Corynebacterium diphtheria (diphtheria);cholera; B. anthracis (anthrax); donovanosis (granuloma inguinale); andbartonellosis. Diseases caused by pathogenic anaerobic bacteria includetetanus; botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever, Rocky Mountain spottedfever, Q fever, and Rickettsialpox. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes encompass pathogenic protozoans and helminths and infectionsproduced thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans;Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm)infections.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA), as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communes and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the vectors of the invention to deliver immunogensagainst the variable region of the T cells elicit an immune responseincluding CTLs to eliminate those T cells. In RA, several specificvariable regions of TCRs which are involved in the disease have beencharacterized. These TCRs include V-3, V-14, V-17 and Vα-17. Thus,delivery of a nucleic acid sequence that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in RA. In MS, several specific variable regions of TCRs whichare involved in the disease have been characterized. These TCRs includeV-7 and Vα-10. Thus, delivery of a nucleic acid sequence that encodes atleast one of these polypeptides will elicit an immune response that willtarget T cells involved in MS. In scleroderma, several specific variableregions of TCRs which are involved in the disease have beencharacterized. These TCRs include V-6, V-8, V-14 and Vα-16, Vα-3C, Vα-7,Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12. Thus, delivery of a chimericadenovirus that encodes at least one of these polypeptides will elicitan immune response that will target T cells involved in scleroderma.

C. Ad-Mediated Delivery Methods

The therapeutic levels, or levels of immunity, of the selected gene canbe monitored to determine the need, if any, for boosters. Following anassessment of CD8+ T cell response, or optionally, antibody titers, inthe serum, optional booster immunizations may be desired. Optionally,the adenoviral vectors of the invention may be delivered in a singleadministration or in various combination regimens, e.g., in combinationwith a regimen or course of treatment involving other active ingredientsor in a prime-boost regimen. A variety of such regimens have beendescribed in the art and may be readily selected.

For example, prime-boost regimens may involve the administration of aDNA (e.g., plasmid) based vector to prime the immune system to a secondor further, booster, administration with a traditional antigen, such asa protein or a recombinant virus carrying the sequences encoding such anantigen. See, e.g., WO 00/11140, published Mar. 2, 2000, incorporated byreference. Alternatively, an immunization regimen may involve theadministration of a chimeric adenoviral vector of the invention to boostthe immune response to a vector (either viral or DNA-based) carrying anantigen, or a protein. In still another alternative, an immunizationregimen involves administration of a protein followed by booster with avector encoding the antigen.

In one embodiment, the invention provides a method of priming andboosting an immune response to a selected antigen by delivering aplasmid DNA vector carrying said antigen, followed by boosting with anadenoviral vector of the invention. In one embodiment, the prime-boostregimen involves the expression of multiproteins from the prime and/orthe boost vehicle. See, e.g., R. R. Amara, Science, 292:69-74 (6 Apr.2001) which describes a multiprotein regimen for expression of proteinsubunits useful for generating an immune response against HIV and SIV.For example, a DNA prime may deliver the Gag, Pol, Vif, VPX and Vpr andEnv, Tat, and Rev from a single transcript. Alternatively, the SIV Gag,Pol and HIV-1 Env is delivered in a recombinant adenovirus construct ofthe invention. Still other regimens are described in WO 99/16884 and WO01/54719.

However, the prime-boost regimens are not limited to immunization forHIV or to delivery of these antigens. For example, priming may involvedelivering with a first vector of the invention followed by boostingwith a second vector, or with a composition containing the antigenitself in protein form. In one example, the prime-boost regimen canprovide a protective immune response to the virus, bacteria or otherorganism from which the antigen is derived. In another desiredembodiment, the prime-boost regimen provides a therapeutic effect thatcan be measured using convention assays for detection of the presence ofthe condition for which therapy is being administered.

The priming composition may be administered at various sites in the bodyin a dose dependent manner, which depends on the antigen to which thedesired immune response is being targeted. The invention is not limitedto the amount or situs of injection(s) or to the pharmaceutical carrier.Rather, the regimen may involve a priming and/or boosting step, each ofwhich may include a single dose or dosage that is administered hourly,daily, weekly or monthly, or yearly. As an example, the mammals mayreceive one or two doses containing between about 10 μg to about 50 μgof plasmid in carrier. A desirable amount of a DNA composition rangesbetween about 1 μg to about 10,000 μg of the DNA vector. Dosages mayvary from about 1 μg to 1000 μg DNA per kg of subject body weight. Theamount or site of delivery is desirably selected based upon the identityand condition of the mammal. The dosage unit of the vector suitable fordelivery of the antigen to the mammal is described herein. The vector isprepared for administration by being suspended or dissolved in apharmaceutically or physiologically acceptable carrier such as isotonicsaline; isotonic salts solution or other formulations that will beapparent to those skilled in such administration. The appropriatecarrier will be evident to those skilled in the art and will depend inlarge part upon the route of administration. The compositions of theinvention may be administered to a mammal according to the routesdescribed above, in a sustained release formulation using abiodegradable biocompatible polymer, or by on-site delivery usingmicelles, gels and liposomes. Optionally, the priming step of thisinvention also includes administering with the priming composition, asuitable amount of an adjuvant, such as are defined herein.

Preferably, a boosting composition is administered about 2 to about 27weeks after administering the priming composition to the mammaliansubject. The administration of the boosting composition is accomplishedusing an effective amount of a boosting composition containing orcapable of delivering the same antigen as administered by the primingDNA vaccine. The boosting composition may be composed of a recombinantviral vector derived from the same viral source (e.g., adenoviralsequences of the invention) or from another source. Alternatively, the“boosting composition” can be a composition containing the same antigenas encoded in the priming DNA vaccine, but in the form of a protein orpeptide, which composition induces an immune response in the host. Inanother embodiment, the boosting composition contains a DNA sequenceencoding the antigen under the control of a regulatory sequencedirecting its expression in a mammalian cell, e.g., vectors such aswell-known bacterial or viral vectors. The primary requirements of theboosting composition are that the antigen of the composition is the sameantigen, or a cross-reactive antigen, as that encoded by the primingcomposition.

In another embodiment, the adenoviral vectors of the invention are alsowell suited for use in a variety of other immunization and therapeuticregimens. Such regimens may involve delivery of adenoviral vectors ofthe invention simultaneously or sequentially with Ad vectors ofdifferent serotype capsids, regimens in which adenoviral vectors of theinvention are delivered simultaneously or sequentially with non-Advectors, regimens in which the adenoviral vectors of the invention aredelivered simultaneously or sequentially with proteins, peptides, and/orother biologically useful therapeutic or immunogenic compounds. Suchuses will be readily apparent to one of skill in the art.

IV. Simian Adenovirus 18 Sequences

The invention provides nucleic acid sequences and amino acid sequencesof Ad SA18, which are isolated from the other viral material with whichthey are associated in nature. These sequences are useful in preparingheterologous molecules containing the nucleic acid sequences and aminoacid sequences, and regions or fragments thereof as are describedherein, viral vectors which are useful for a variety of purposes,including the constructs and compositions, and such methods as aredescribed herein for the chimeric adenoviruses, including, e.g., in hostcells for production of viruses requiring adenoviral helper functions,as delivery vehicles for heterologous molecules such as those describedherein. These sequences are also useful in generating the chimericadenoviruses of the invention.

A. Nucleic Acid Sequences

The SA18 nucleic acid sequences of the invention include nucleotides SEQD NO: 12, nt 1 to 31967. See, Sequence Listing, which is incorporated byreference herein. The nucleic acid sequences of the invention furtherencompass the strand which is complementary to the sequences of SEQ IDNO: 12, as well as the RNA and cDNA sequences corresponding to thesequences of these sequences figures and their complementary strands.Further included in this invention are nucleic acid sequences which aregreater than 95 to 98%, and more preferably about 99 to 99.9% homologousor identical to the Sequence Listing. Also included in the nucleic acidsequences of the invention are natural variants and engineeredmodifications of the sequences provided in SEQ ID NO: 12 and theircomplementary strands. Such modifications include, for example, labelsthat are known in the art, methylation, and substitution of one or moreof the naturally occurring nucleotides with a degenerate nucleotide.

The invention further encompasses fragments of the sequences of SA18,their complementary strand, cDNA and RNA complementary thereto. Suitablefragments are at least 15 nucleotides in length, and encompassfunctional fragments, i.e., fragments which are of biological interest.For example, a functional fragment can express a desired adenoviralproduct or may be useful in production of recombinant viral vectors.Such fragments include the gene sequences and fragments listed in thetables below.

The following tables provide the transcript regions and open readingframes in the simian adenovirus sequences of the invention. For certaingenes, the transcripts and open reading frames (ORFs) are located on thestrand complementary to that presented in SEQ ID NO: 12. See, e.g., E2b,E4 and E2a. The calculated molecular weights of the encoded proteins arealso shown. Adenovirus Ad SA18, Gene SEQ ID NO: 12 Region Protein startEnd M.W. ITR 1 180 E1a 13S 916 1765 27264 12S 916 1765 24081 E1b Small T1874 2380 19423 LargeT 2179 3609 52741 IX 3678 4079 13701 E2b IVa2 54784126 51295 Polymerase 13745 5229 128392 PTP 13745 8597 75358 Agnoprotein8007 8705 23610 L1 52/55 kD 10788 11945 43416 IIIa 11966 13699 63999 L2Penton 13796 15322 57166 VII 15328 15873 20352 V 15920 17050 42020 L3 VI17348 18154 29222 Hexon 18257 21010 102912 Endoprotease 21029 2164023015 2a DBP 23147 21711 53626 L4 100 kD 23175 25541 87538 22 kD 2520425797 22206 homolog 33 kD 25204 26025 24263 homolog VIII 26107 2681725490 E3 Orf#1 26817 27125 11814 L5 Fiber 27192 29015 65455 E4 Orf 6/730169 29067 13768 Orf 6 30169 29303 33832 Orf 4 30464 30099 14154 Orf 330816 30466 13493 Orf 2 31205 30813 14698 Orf 1 31608 31231 14054 ITR31788 31967

The SA18 adenoviral nucleic acid sequences are useful as therapeutic andimmunogenic agents and in construction of a variety of vector systemsand host cells. Such vectors are useful for any of the purposesdescribed above for the chimeric adenovirus. Additionally, these SA18sequences and products may be used alone or in combination with otheradenoviral sequences or fragments, or in combination with elements fromother adenoviral or non-adenoviral sequences. The adenoviral sequencesof the invention are also useful as antisense delivery vectors, genetherapy vectors, or vaccine vectors, and in methods of using same. Thus,the invention further provides nucleic acid molecules, gene deliveryvectors, and host cells which contain the Ad sequences of the invention.

For example, the invention encompasses a nucleic acid moleculecontaining simian Ad ITR sequences of the invention. In another example,the invention provides a nucleic acid molecule containing simian Adsequences of the invention encoding a desired Ad gene product. Stillother nucleic acid molecule constructed using the sequences of theinvention will be readily apparent to one of skill in the art, in viewof the information provided herein.

In one embodiment, the simian Ad gene regions identified herein may beused in a variety of vectors for delivery of a heterologous molecule toa cell. Examples of such molecules and methods of delivery are providedin Section III herein. For example, vectors are generated for expressionof an adenoviral capsid protein (or fragment thereof) for purposes ofgenerating a viral vector in a packaging host cell. Such vectors may bedesigned for expression in trans. Alternatively, such vectors aredesigned to provide cells which stably contain sequences which expressdesired adenoviral functions, e.g., one or more of E1a, E1b, theterminal repeat sequences, E2a, E2b, E4, E40RF6 region.

In addition, the adenoviral gene sequences and fragments thereof areuseful for providing the helper functions necessary for production ofhelper-dependent viruses (e.g., adenoviral vectors deleted of essentialfunctions or adeno-associated viruses (AAV)). For such productionmethods, the simian adenoviral sequences of the invention are utilizedin such a method in a manner similar to those described for the humanAd. However, due to the differences in sequences between the simianadenoviral sequences of the invention and those of human Ad, the use ofthe sequences of the invention essentially eliminate the possibility ofhomologous recombination with helper functions in a host cell carryinghuman Ad E1 functions, e.g., 293 cells, which may produce infectiousadenoviral contaminants during rAAV production.

Methods of producing rAAV using adenoviral helper functions have beendescribed at length in the literature with human adenoviral serotypes.See, e.g., U.S. Pat. No. 6,258,595 and the references cited therein.See, also, U.S. Pat. No. 5,871,982; WO 99/14354; WO 99/15685; WO99/47691. These methods may also be used in production of non-humanserotype AAV, including non-human primate AAV serotypes. The simianadenoviral gene sequences of the invention which provide the necessaryhelper functions (e.g., E1a, E1b, E2a and/or E4 ORF6) can beparticularly useful in providing the necessary adenoviral function whileminimizing or eliminating the possibility of recombination with anyother adenoviruses present in the rAAV-packaging cell which aretypically of human origin. Thus, selected genes or open reading framesof the adenoviral sequences of the invention may be utilized in theserAAV production methods.

Alternatively, recombinant adenoviral simian vectors of the inventionmay be utilized in these methods. Such recombinant adenoviral simianvectors may include, e.g., a hybrid simian Ad/AAV in which simian Adsequences flank a rAAV expression cassette composed of, e.g., AAV 3′and/or 5′ ITRs and a transgene under the control of regulatory sequenceswhich control its expression. One of skill in the art will recognizethat still other simian adenoviral vectors and/or gene sequences of theinvention will be useful for production of rAAV and other virusesdependent upon adenoviral helper.

In still another embodiment, nucleic acid molecules are designed fordelivery and expression of selected adenoviral gene products in a hostcell to achieve a desired physiologic effect. For example, a nucleicacid molecule containing sequences encoding an adenovirus E1a protein ofthe invention may be delivered to a subject for use as a cancertherapeutic. Optionally, such a molecule is formulated in a lipid-basedcarrier and preferentially targets cancer cells. Such a formulation maybe combined with other cancer therapeutics (e.g., cisplatin, taxol, orthe like). Still other uses for the adenoviral sequences provided hereinwill be readily apparent to one of skill in the art.

In addition, one of skill in the art will readily understand that the Adsequences of the invention can be readily adapted for use for a varietyof viral and non-viral vector systems for in vitro, ex vivo or in vivodelivery of therapeutic and immunogenic molecules, including any ofthose identified as being deliverable via the chimeric adenoviruses ofthe invention. For example, the simian Ad genome of the invention can beutilized in a variety of rAd and non-rAd vector systems. Such vectorssystems may include, e.g., plasmids, lentiviruses, retroviruses,poxviruses, vaccinia viruses, and adeno-associated viral systems, amongothers. Selection of these vector systems is not a limitation of thepresent invention.

The invention further provides molecules useful for production of thesimian and simian-derived proteins of the invention. Such moleculeswhich carry polynucleotides including the simian Ad DNA sequences of theinvention can be in the form of a vector.

B. Simian Adenoviral Proteins of the Invention

The invention further provides gene products of the above adenoviruses,such as proteins, enzymes, and fragments thereof, which are encoded bythe adenoviral nucleic acids of the invention. The invention furtherencompasses SA18 proteins, enzymes, and fragments thereof, having theamino acid sequences encoded by these nucleic acid sequences which aregenerated by other methods. Such proteins include those encoded by theopen reading frames identified in the tables above, and fragmentsthereof.

Thus, in one aspect, the invention provides unique simian adenoviralproteins which are substantially pure, i.e., are free of other viral andproteinaceous proteins. Preferably, these proteins are at least 10%homogeneous, more preferably 60% homogeneous, and most preferably 95%homogeneous.

In one embodiment, the invention provides unique simian-derived capsidproteins. As used herein, a simian-derived capsid protein includes anyadenoviral capsid protein that contains a SA18 capsid protein or afragment thereof, as defined above, including, without limitation,chimeric capsid proteins, fusion proteins, artificial capsid proteins,synthetic capsid proteins, and recombinantly capsid proteins, withoutlimitation to means of generating these proteins.

Suitably, these simian-derived capsid proteins contain one or more SA18regions or fragments thereof (e.g., a hexon, penton, fiber or fragmentthereof) in combination with capsid regions or fragments thereof ofdifferent adenoviral serotypes, or modified simian capsid proteins orfragments, as described herein. A “modification of a capsid proteinassociated with altered tropism” as used herein includes an alteredcapsid protein, i.e, a penton, hexon or fiber protein region, orfragment thereof, such as the knob domain of the fiber region, or apolynucleotide encoding same, such that specificity is altered. Thesimian-derived capsid may be constructed with one or more of the simianAd of the invention or another Ad serotypes which may be of human ornon-human origin. Such Ad may be obtained from a variety of sourcesincluding the ATCC, commercial and academic sources, or the sequences ofthe Ad may be obtained from GenBank or other suitable sources.

The amino acid sequences of the simian adenoviruses penton proteins ofthe invention are provided herein. The AdSA18 penton protein is providedin SEQ ID NO: 13. Suitably, any of these penton proteins, or uniquefragments thereof, may be utilized for a variety of purposes. Examplesof suitable fragments include the penton having N-terminal and/orC-terminal truncations of about 50, 100, 150, or 200 amino acids, basedupon the amino acid numbering provided above. Other suitable fragmentsinclude shorter internal, C-terminal, or N-terminal fragments, Further,the penton protein may be modified for a variety of purposes known tothose of skill in the art.

The invention further provides the amino acid sequences of the hexonprotein of SA18, SEQ ID NO:14. Suitably, this hexon protein, or uniquefragments thereof, may be utilized for a variety of purposes. Examplesof suitable fragments include the hexon having N-terminal and/orC-terminal truncations of about 50, 100, 150, 200, 300, 400, or 500amino acids, based upon the amino acid numbering provided above and inSEQ ID NO: 14. Other suitable fragments include shorter internal,C-terminal, or N-terminal fragments. For example, one suitable fragmentthe loop region (domain) of the hexon protein, designated DE1 and FG1,or a hypervariable region thereof. Such fragments include the regionsspanning amino acid residues about 125 to 443; about 138 to 441, orsmaller fragments, such as those spanning about residue 138 to residue163; about 170 to about 176; about 195 to about 203; about 233 to about246; about 253 to about 264; about 287 to about 297; about 404 to about430, about 430 to 550, about 545 to 650; of the simian hexon proteins,with reference to SEQ ID NO: 14. Other suitable fragments may be readilyidentified by one of skill in the art. Further, the hexon protein may bemodified for a variety of purposes known to those of skill in the art.Because the hexon protein is the determinant for serotype of anadenovirus, such artificial hexon proteins would result in adenoviruseshaving artificial serotypes. Other artificial capsid proteins can alsobe constructed using the chimp Ad penton sequences and/or fibersequences of the invention and/or fragments thereof.

In one example, it may be desirable to generate an adenovirus having analtered hexon protein utilizing the sequences of a hexon protein of theinvention. One suitable method for altering hexon proteins is describedin U.S. Pat. No. 5,922,315, which is incorporated by reference. In thismethod, at least one loop region of the adenovirus hexon is changed withat least one loop region of another adenovirus serotype. Thus, at leastone loop region of such an altered adenovirus hexon protein is a simianAd hexon loop region of the invention. In one embodiment, a loop regionof the SA18 hexon protein is replaced by a loop region from anotheradenovirus serotype. In another embodiment, the loop region of the SA18hexon is used to replace a loop region from another adenovirus serotype.Suitable adenovirus serotypes may be readily selected from among humanand non-human serotypes, as described herein. SA18 is selected forpurposes of illustration only; the other simian Ad hexon proteins of theinvention may be similarly altered, or used to alter another Ad hexon.The selection of a suitable serotype is not a limitation of the presentinvention. Still other uses for the hexon protein sequences of theinvention will be readily apparent to those of skill in the art.

The invention further encompasses the fiber proteins of the simianadenoviruses of the invention. The fiber protein of AdSA18 has the aminoacid sequence of SEQ ID NO: 15. Suitably, this fiber protein, or uniquefragments thereof, may be utilized for a variety of purposes. Onesuitable fragment is the fiber knob, which spans about amino acids 247to 425 of SEQ ID NO: 15. Examples of other suitable fragments includethe fiber having N-terminal and/or C-terminal truncations of about 50,100, 150, or 200 amino acids, based upon the amino acid numberingprovided above and in SEQ ID NO: 15. Still other suitable fragmentsinclude internal fragments. Further, the fiber protein may be modifiedusing a variety of techniques known to those of skill in the art.

The invention further encompasses unique fragments of the proteins ofthe invention which are at least 8 amino acids in length. However,fragments of other desired lengths can be readily utilized. In addition,the invention encompasses such modifications as may be introduced toenhance yield and/or expression of an SA18 gene product, e.g.,construction of a fusion molecule in which all or a fragment of the SA18gene product is fused (either directly or via a linker) with a fusionpartner to enhance. Other suitable modifications include, withoutlimitation, truncation of a coding region (e.g., a protein or enzyme) toeliminate a pre- or pro-protein ordinarily cleaved and to provide themature protein or enzyme and/or mutation of a coding region to provide asecretable gene product. Still other modifications will be readilyapparent to one of skill in the art. The invention further encompassesproteins having at least about 95% to 99% identity to the SA18 proteinsprovided herein.

As described herein, vectors of the invention containing the adenoviralcapsid proteins of the invention are particularly well suited for use inapplications in which the neutralizing antibodies diminish theeffectiveness of other Ad serotype based vectors, as well as other viralvectors. The rAd vectors of the invention are particularly advantageousin readministration for repeat gene therapy or for boosting immuneresponse (vaccine titers). Examples of such regimens are providedherein.

Under certain circumstances, it may be desirable to use one or more ofthe SA18 gene products (e.g., a capsid protein or a fragment thereof) togenerate an antibody. The term “an antibody,” as used herein, refers toan immunoglobulin molecule which is able to specifically bind to anepitope. Thus, the antibodies of the invention bind, preferablyspecifically and without cross-reactivity, to a SA18 epitope. Theantibodies in the present invention exist in a variety of formsincluding, for example, high affinity polyclonal antibodies, monoclonalantibodies, synthetic antibodies, chimeric antibodies, recombinantantibodies and humanized antibodies. Such antibodies originate fromimmunoglobulin classes IgG, IgM, IgA, IgD and IgE.

Such antibodies may be generated using any of a number of methods knowin the art. Suitable antibodies may be generated by well-knownconventional techniques, e.g. Kohler and Milstein and the many knownmodifications thereof. Similarly desirable high titer antibodies aregenerated by applying known recombinant techniques to the monoclonal orpolyclonal antibodies developed to these antigens [see, e.g., PCT PatentApplication No. PCT/GB85/00392; British Patent Application PublicationNo. GB2188638A; Amit et al., 1986 Science, 233:747-753; Queen et al.,1989 Proc. Natl. Acad. Sci. USA, 86:10029-10033; PCT Patent ApplicationNo. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988);Huse et al, 1988a Science, 246:1275-1281]. Alternatively, antibodies canbe produced by manipulating the complementarity determining regions ofanimal or human antibodies to the antigen of this invention. See, e.g.,E. Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4,The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology ofMonoclonal Antibodies, Springer-Verlag (June, 1994); Harlow et al.,1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; and Bird et al., 1988, Science 242:423-426.Further provided by the present invention are anti-idiotype antibodies(Ab2) and anti-anti-idiotype antibodies (Ab3). See, e.g., M. Wettendorffet al., “Modulation of anti-tumor immunity by anti-idiotypicantibodies.” In Idiotypic Network and Diseases, ed. by J. Cerny and J.Hiernaux, 1990 J. Am. Soc. Microbiol., Washington D.C.: pp. 203-229].These anti-idiotype and anti-anti-idiotype antibodies are produced usingtechniques well known to those of skill in the art. These antibodies maybe used for a variety of purposes, including diagnostic and clinicalmethods and kits.

Under certain circumstances, it may be desirable to introduce adetectable label or a tag onto a SA18 gene product, antibody or otherconstruct of the invention. As used herein, a detectable label is amolecule which is capable, alone or upon interaction with anothermolecule, of providing a detectable signal. Most desirably, the label isdetectable visually, e.g. by fluorescence, for ready use inimmunohistochemical analyses or immunofluorescent microscopy. Forexample, suitable labels include fluorescein isothiocyanate (FITC),phycoerythrin (PE), allophycocyanin (APC), coriphosphine-O(CPO) ortandem dyes, PE-cyanin-5 (PC5), and PE-Texas Red (ECD). All of thesefluorescent dyes are commercially available, and their uses known to theart. Other useful labels include a colloidal gold label. Still otheruseful labels include radioactive compounds or elements. Additionally,labels include a variety of enzyme systems that operate to reveal acolorimetric signal in an assay, e.g., glucose oxidase (which usesglucose as a substrate) releases peroxide as a product which in thepresence of peroxidase and a hydrogen donor such as tetramethylbenzidine (TMB) produces an oxidized TMB that is seen as a blue color.Other examples include horseradish peroxidase (HRP) or alkalinephosphatase (AP), and hexokinase in conjunction with glucose-6-phosphatedehydrogenase which reacts with ATP, glucose, and NAD+ to yield, amongother products, NADH that is detected as increased absorbance at 340 nmwavelength.

Other label systems that are utilized in the methods of this inventionare detectable by other means, e.g., colored latex microparticles [BangsLaboratories, Indiana] in which a dye is embedded are used in place ofenzymes to form conjugates with the target sequences provide a visualsignal indicative of the presence of the resulting complex in applicableassays.

Methods for coupling or associating the label with a desired molecule resimilarly conventional and known to those of skill in the art. Knownmethods of label attachment are described [see, for example, Handbook ofFluorescent probes and Research Chemicals, 6th Ed., R. P. M. Haugland,Molecular Probes, Inc., Eugene, Oreg., 1996; Pierce Catalog andHandbook, Life Science and Analytical Research Products, Pierce ChemicalCompany, Rockford, Ill., 1994/1995]. Thus, selection of the label andcoupling methods do not limit this invention.

The sequences, proteins, and fragments of the invention may be producedby any suitable means, including recombinant production, chemicalsynthesis, or other synthetic means. Suitable production techniques arewell known to those of skill in the art. See, e.g., Sambrook et al,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (ColdSpring Harbor, N.Y.). Alternatively, peptides can also be synthesized bythe well known solid phase peptide synthesis methods (Merrifield, J. Am.Chem. Soc., 85:2149 (1962); Stewart and Young, Solid Phase PeptideSynthesis (Freeman, San Francisco, 1969) pp. 27-62). These and othersuitable production methods are within the knowledge of those of skillin the art and are not a limitation of the present invention.

In addition, one of skill in the art will readily understand that the Adsequences of the invention can be readily adapted for use for a varietyof viral and non-viral vector systems for in vitro, ex vivo or in vivodelivery of therapeutic and immunogenic molecules. For example, in oneembodiment, the simian Ad capsid proteins and other simian adenovirusproteins described herein are used for non-viral, protein-based deliveryof genes, proteins, and other desirable diagnostic, therapeutic andimmunogenic molecules. In one such embodiment, a protein of theinvention is linked, directly or indirectly, to a molecule for targetingto cells with a receptor for adenoviruses. Preferably, a capsid proteinsuch as a hexon, penton, fiber or a fragment thereof having a ligand fora cell surface receptor is selected for such targeting. Suitablemolecules for delivery are selected from among the therapeutic moleculesdescribed herein and their gene products. A variety of linkersincluding, lipids, polyLys, and the like may be utilized as linkers. Forexample, the simian penton protein may be readily utilized for such apurpose by production of a fusion protein using the simian pentonsequences in a manner analogous to that described in Medina-Kauwe L K,et al, Gene Ther. 2001 May; 8(10):795-803 and Medina-Kauwe L K, et al,Gene Ther. 2001 December; 8(23): 1753-1761. Alternatively, the aminoacid sequences of simian Ad protein IX may be utilized for targetingvectors to a cell surface receptor, as described in US Patent Appln20010047081. Suitable ligands include a CD40 antigen, an RGD-containingor polylysine-containing sequence, and the like. Still other simian Adproteins, including, e.g., the hexon protein and/or the fiber protein,may be used for used for these and similar purposes.

Still other adenoviral proteins of the invention may be used as alone,or in combination with other adenoviral protein, for a variety ofpurposes which will be readily apparent to one of skill in the art. Inaddition, still other uses for the adenoviral proteins of the inventionwill be readily apparent to one of skill in the art.

The compositions of this invention include vectors that deliver aheterologous molecule to cells, either for therapeutic or vaccinepurposes. Such vectors, containing simian adenovirus DNA of SA18 and aminigene, can be constructed using techniques such as those describedherein for the chimeric adenoviruses and such techniques as are known inthe art. Alternatively, SA19 may be a source for sequences of thechimeric adenoviruses are described herein.

The following examples illustrate construction and use of severalchimeric viruses, including Pan5/C1, hu5/Pan7 and hu5/SV25, andPan6/Pan7. However, these chimera are illustrative only and are notintended to limit the invention to those illustrated embodiments.

EXAMPLE 1 Construction of Pan5/C1 Chimeric Simian Viruses

Five different adenoviruses initially isolated from the chimpanzee,AdC68 [U.S. Pat. No. 6,083,716], AdPan5, AdPan7, AdPan6 and AdC1 [U.S.Pat. No. 6,083,716] have been sequenced. See, International ApplicationNo. PCT/US02/33645, filed November 2002 for the sequences of Pan5 [SEQID NO: 1], Pan7 [SEQ ID NO:3], and Pan6 [SEQ ID NO:2]. This applicationalso provides sequences for SV1, SV25 and SV39 [SEQ ID No. 4, 5, 6,respectively]. Sequence comparison of the capsid protein sequencespredicted that AdC1 clearly belonged to a different serological subgroupthan the other four chimpanzee derived adenoviruses.

However, attempts to cultivate AdC1 in HEK293 cells revealed it to befastidious in its growth characteristics (data not shown) and thereforepossibly unsuitable for use as a vector using the currently available E1complementing cell lines. However, because of the obvious sequencedissimilarity of AdC1 capsid protein sequence from the other chimpanzeederived adenoviruses (as well as the huAd5), chimeric adenovirus vectorswere generated with the capsid characteristics of AdC1. In view of theabove-mentioned drawbacks associated with only making hexon changes,more extensive replacements were made in the chimera described herein,i.e., construction of chimeras where the replacement went beyond justthe hexon, to achieve two goals. The first was to determine whethermaking extended replacements would allow for the rescue of virusescontaining hexons of unrelated serotypes that may not otherwise beamenable to rescue. The second goal was to test whether the growthcharacteristics of adenovirus vectors such as AdPan5, that have beenfound in our laboratory to be able to be grown to high titer for thepurpose of manufacture, would also be present in the chimeric virus,particularly when the hexon (and other capsid proteins) are derived froma virus such as AdC1 that are difficult to grow to a high yield in celllines such as HEK293. An added bonus of extending the replacement toinclude the fiber protein would be to further increase the antigenicdissimilarity to beyond that afforded by a hexon change alone.

As an alternative to obtaining purified virus as source for adenoviralDNA to sequence, we have resorted to cloning restriction fragments ofviral DNA obtained from infected cells (“Hirt prep”). The firstadenovirus we have sequenced in this way is Simian Adenovirus. EcoRIdigestion of the Simian Adenovirus yielded 7 fragments. Shotgun cloningyielded clones of the 5 internal fragments, which were cloned andsequenced. Completion of the sequencing was carried out by walkingtowards each of the ends of the genome. The map of the genome is shownin FIG. 1.

A. Construction of Two Pan5/C1 Chimeric Plasmids

The overall approach towards constructing chimeric viruses was to firstassemble the complete E1 deleted virus DNA into a single plasmid flankedby recognition sites for the restriction enzyme SwaI, digest the plasmidDNA with SwaI to release the virus DNA ends, and transfect the DNA intoHEK293 cells to determine whether viable chimeric adenovirus could berescued. Two chimeric virus plasmids were constructed, p5C1short andp5C1long.

The plasmid p5C1short harbors an E1 deleted Pan5 virus where an internal15226 bp segment (18332-33557) has been replaced by a functionallyanalogous 14127 bp (18531-32657) from AdC1. This results in thereplacement of the Pan5 proteins hexon, endoprotease, DNA bindingprotein, 100 kD scaffolding protein, 33 kD protein, protein VIII, andfiber, as well as the entire E3 region, with the homologous segment fromAdC1. The ClaI site at the left end of the AdC1 fragment is at thebeginning of the hexon gene and the resulting protein is identical tothe C1 hexon. The EcoRI site which constitutes the right end of the AdC1fragment is within the E4 orf 7 part of the AdC1. The right end wasligated to a PCR generated right end fragment from AdPan5 such that theregenerated orf 7-translation product is chimeric between AdPan5 andAdC1.

The plasmid p5C1 long harbors an E1 deleted Pan5 virus where an internal25603 bp segment (7955-33557) has been replaced by a functionallyanalogous 24712 bp (7946-32657) from AdC1. This results in thereplacement of the AdPan5 pre-terminal protein, 52/55 kD protein, pentonbase protein, protein VII, Mu, and protein VI with those from AdC1 inaddition to those replaced in p5C1 short. The AscI site at the left endof the AdC1 fragment is at the beginning of the DNA polymerase gene andresults in a chimeric protein where the first 165 amino acids of theAdPan5 DNA polymerase has been replaced by a 167 amino acid segment fromAdC1 DNA polymerase. In this N-terminal region, the homology between theAdPan5 and AdC1 DNA polymerase proteins is 81% (72% identity).

The plasmid pDVP5Mlu which contains the left end of AdPan5 was used asthe starting plasmid for the chimeric vector construction.

The plasmid pDVP5Mlu was made as follows. A synthetic DNA fragmentharboring recognition sites for the restriction enzymes SmaI, MluI,EcoRI and EcoRV respectively was ligated into pBR322 digested with EcoRIand NdeI so as to retain the origin of replication and thebeta-lactamase gene. The left end of Pan5 extending to the MluI site(15135 bp) was cloned into this plasmid between the SmaI and MluI sites.The E1 gene was functionally deleted and replaced by a DNA fragmentharboring recognition sites for the extremely rare cutter restrictionenzyme sites I-CeuI and PI-SceI). The 2904 base pairs of the right endof Pan-5 was PCR amplified using the primers P5L [GCG CAC GCG TCT CTATCG ATG AAT TCC ATT GGT GAT GGA CAT GC, SEQ ID NO:7] and P51TR [GCG CATTTA AAT CAT CAT CAA TAA TAT ACC TCA AAC, SEQ ID NO:8] using Tgopolymerase (Roche). The PCR product was cut with MluI and SwaI, andcloned between MluI and EcoRV of pDVP5Mlu to yield pPan5Mlu+RE. A 3193bp fragment extending from the MluI site (15135) to the ClaI (18328)site of Pan5 was then inserted between the same sites of pPan5Mlu+RE toyield pPan5Cla+RE. The 3671 bp ClaI (18531) to EcoRI (22202) fragment ofthe adenovirus C1 was cloned into pPan5Cla+RE between ClaI (16111) andEcoRI (16116) to yield pPan5C1delRI. The 10452 bp internal EcoRIfragment of the adenovirus C1 (22202-32653) was cloned into the EcoRIsite of pPan5C1delRI to yield p5C1short. To construct pSC1long, the AdC1replacement was further extended by replacing the AscI-ClaI 10379 bpfragment of AdPan5 in p5C1short with the AdC1 AscI-ClaI 10591 bpfragment. Finally a green fluorescent protein (GFP) expression cassettewas inserted into both p5C1short and p5C1long between the I-CeuI andPI-SceI sites to yield p5C1shortGFP and p5C1longGFP respectively.

B. Rescue of Chimeric Pan5/C1 Recombinant Vector Adenoviruses

The plasmids p5C1shortGFP and p5C1longGFP were digested with therestriction enzyme SwaI and transfected into HEK 293 cells. A typicaladenovirus induced cytopathic effect was observed. The rescue ofrecombinant chimeric adenovirus from the p5C1longGFP transfection wasconfirmed by collecting the supernatant from the transfection andre-infecting fresh cells which were found to be transduced as determinedby GFP expression. Viral DNA prepared from the chimeric recombinantvirus was digested with several restriction enzymes and found to havethe expected pattern on electrophoresis (data not shown).

The chimeric adenoviral construct with the shorter replacement p5C1shortencodes the C1 proteins hexon and fiber as well as the intervening openreading frames for endoprotease, DNA binding protein, 100 kDascaffolding protein, 33 kDa protein, and protein VIII. (The E3 region isalso included within this region but is unlikely to impact on theviability of the chimeric virus). When the replacement was extended toinclude the additional AdC1 proteins pTP (pre-terminal protein), 52/55kDa protein, penton base, protein VII, Mu, and protein VI, there was nodifficulty in rescuing viable chimeric virus. In this experiment, thechimeric adenovirus construction strategy utilized the presence of AscIand ClaI restriction enzyme sites present on the genes for DNApolymerase and hexon respectively on both AdPan5 and AdC1.

The reasons for the relatively higher yield of the chimeric viruscompared to the wild-type AdC1 virus are not clear. In the growth of the5C1 chimeric virus in 293 cells, the adenoviral early region geneproducts of E1 and E4 are derived from Ad5 and AdPan5 respectively. TheE1 and E4 gene products bind, regulate and de-repress several cellulartranscription complexes and coordinate their activity towards viralmultiplication. Thus it is possible that the E1 gene products suppliedin trans from the 293 cells and the E4 gene products from AdPan5 aremore optimal in the human 293 cell background than are the equivalentAdC1 gene products. This may also apply to the activity of the majorlate promoter whose activity is responsible for the transcription of thecapsid protein genes. In the chimeric virus, the major late promoter,and the protein IVa2 which transactivates it, are derived from AdPan5.However the E2 gene products required for adenoviral DNA replication pTPand single-stranded DNA—binding protein are derived from AdC1. Theadenoviral DNA polymerase, which complexes with pTP, is chimeric inAd5C1 but mostly AdPan5 derived.

EXAMPLE 2 Construction of Ad5 Chimeric Simian Viruses

Plasmids have been constructed where the structural proteins derive fromthe chimpanzee adenovirus Pan 7 and the flanking sequences are derivedfrom human Ad5 (the commonly used vector strain). The Adhu5-Pan7chimeric adenovirus has been rescued, demonstrating that the chimericvirus construction method used to derive the chimeric virus is broadlyapplicable.

A. Construction of the Ad5-Pan 7 Chimeric Adenovirus

A plasmid was constructed which harbors the complete (E1 deleted)chimeric genome in order to establish that the chimeric adenovirus isviable, and then transfected the plasmid into the E1 complementing cellline HEK 293. It was found that the recombinant virus could be rescued.The chimeric adenovirus genome that was constructed is composed of aleft end segment derived from Ad5 that contributes the ITR, the E1deletion region containing the transgene expression cassette, the pIXand IVa2 genes and 954 C-terminal amino acids of the polymerase gene(which is transcribed in the right to left direction from the bottomstrand). Ad5 also contributes the right end of the chimeric genomecontaining the E4 genes and the right ITR. All the other genes presentin the central part of the chimeric construct are derived from thechimpanzee adenovirus Pan 7 including the N-terminal 235 amino acids ofa chimeric DNA polymerase.

In order to construct the plasmid which harbors the complete (E1deleted) chimeric genome, the starting plasmid was pBRAd5lere which iscomprised of three parts; the bacterial origin of replication andampicillin resistance gene derived from the plasmid pBR322, the left endof an Ad5 derived E1 deleted vector extending from the left ITR to theStuI site located at base pair number 5782 of the wild-type Ad5 genome(the E1 deletion extends from base pair 342 to 3533 of the wild-type Ad5genome), and the right end of Ad5 extending from the StuI site at basepair number 31954 of the wild-type Ad5 genome to the right end of theright ITR. The PacI sites located adjacent to the two ITRs are used torelease the Ad5 genome from the bacterial plasmid backbone. The fragmentcontaining I-CeuI and the PI-SceI sites which is located in place of theE1 deletion is used to insert transgene cassettes.

A synthetic DNA oligomer was inserted at the StuI site containing sitesfor AscI, XbaI and EcoRI, which allowed the creation of the plasmidpAd5endsAscRI where using PCR, the Ad5 polymerase gene was extended tobase pair #8068 of the wild-type Ad5 genome and incorporating a newlycreated AscI site at this location by silent mutagenesis of thepolymerase gene (translated from the bottom strand) as depicted below.Original sequence GCG ACG GGC CGA CGC TGC CCG GCT [SEQ ID NO:16] Arg ArgAla Ser [SEQ ID NO:17] Mutated sequence (The AscI recognition site isunderlined) GCG GCG CGC CGA CGC TGC CCG GCT [SEQ ID NO:18] Arg Arg AlaSer [SEQ ID NO: 17]

The Pan 7 fiber containing region was amplified by PCR (mutating thefiber stop codon from TGA to TAA to provide a polyadenylation signalsimilar to that in Ad5) and inserted into the EcoRI site to yieldpAd5endsP7fib. Several cloning steps led to the construction of pH5C₇H₅where the complete chimeric adenoviral genome has been assembled Atransgene cassette expressing GFP (green fluorescent protein) wasinserted between the I-CeuI and PI-SceI sites of pHSC7H5. The finalconstruct was digested with PacI to separate the adenoviral genome fromthe plasmid backbone and transfected into HEK 293 cells. The cell lysatewas harvested 2 weeks later, and the chimeric adenovirus was amplifiedand purified by standard methods.

B. Construction of the Ad5—Simian Virus 25 (SV-25) Chimeric Adenovirus

[N. B. Simian virus 25 (ATCC catalog number VR-201) is distinct from thechimpanzee adenovirus Simian adenovirus 25 ATCC catalog number VR-594]

The construction of the Ad5 based chimeric adenovirus where the left andright end segments are derived from Ad5 and the central portion wasderived from the monkey adenovirus SV-25 was carried out in a mannercompletely analogous to that described above for the chimeric adenovirusdescribed above that is chimeric between Ad5 and the chimpanzeeadenovirus Pan 7. Thus, the chimeric adenovirus genome that wasconstructed is composed of a left end segment derived from Ad5 thatcontributes the ITR, the E1 deletion region containing the transgeneexpression cassette, the pIX and IVa2 genes and 956 C-terminal aminoacids of the polymerase gene. Ad5 also contributes the right end of thechimeric genome containing the E4 genes and the right ITR.[Additionally, the left end of the Ad5 genome was extended beyond thatpresent in pH5C₇H₅ so that 454 base pairs of the Ad5 left end waspresent. Although not absolutely essential, this was done in order toimprove packaging efficiency.] All the genes present in the central partof the chimeric construct are derived from the monkey adenovirus SV-25including the N-terminal 230 amino acids of a chimeric DNA polymerase.The starting plasmid for the construction of the chimeric genome waspAd5endsAscRI which contains both the left and right ends of Ad5 as wellas the created (by silent mutation) AscI site in the polymerase genewhere Ad5-SV25 chimeric fusion was made (as was done for the Ad5-Pan 7chimeric adenovirus). In the final construct pH5S25H5, the SV25 genomesegment has been incorporated by sequential cloning steps, includingcreation of an AscI site at the ligation junction within the polymerasecoding sequence. A transgene cassette expressing GFP (green fluorescentprotein) was inserted between the I-CeuI and PI-SceI sites of pH5S25H5.The final construct was digested with PacI to separate the adenoviralgenome from the plasmid backbone and transfected into HEK 293 cells. Thecell lysate was harvested 2 weeks later, and the chimeric adenovirus wasamplified and purified by standard methods.

FIG. 2 provides the map of the recombinant Adhu5-SV25 chimeric virus.The portion of the genome replaced by DNA from Pan7 is indicated.

EXAMPLE 3 Pan5—C1 Chimeric Vector of Invention as a Delivery Vehicle forImmunogenic Compositions

A Pan 5 (Simian adenovirus 22, a subgroup E adenovirus, also termed C5)—C1 (Simian adenovirus 21, a subgroup B adenovirus) chimeric expressingthe Ebola virus (Zaire) glycoprotein (C5C1C5-CMVGP) was constructed as amodel antigen in order to test the efficacy of the vector C5C1C5-CMVGPas a vaccine; this vector has been compared it to the Adhu5 based vector(H5-CMVGP). Compared to H5-CMVGP, the C5C1-CMVGP vector yielded only aslightly decreased level of GP expression in transduced A549 cells.

Thereafter, GP-specific T cell and B cell responses elicited in B10BRmice vaccinated intramuscularly with either 5×10¹⁰ H5-CMVGP orC5C1-CMVGP vectors were compared.

The C5C1C5-CMVGP vector appeared to induce lower frequencies of gammainterferon producing CD8+ T cells with kinetics slower than the H5-CMVGPvector as determined by intracellular cytokine staining using a H-2krestricted GP-specific peptide as stimulant. The total IgG response toGP, measured by ELISA, was equivalent in serum from mice vaccinated withthe C5C1C5-CMVGP or the H5-CMVGP vectors. However, the C5C1C5-CMVGPvector induced a more potent Th1 type response while the H5-CMVGP vectorstimulated a more balanced Th1/Th2 type response. In a survival study,mice were vaccinated as above and challenged 28 days later with 200LD/50 mouse-adapted Ebola Zaire virus. 100% survival was seen for bothgroups.

EXAMPLE 4 Generation of Chimeric Pan6/Pan7 Vectors

A panel of GFP expressing vectors were generated. This panel includesvectors that are chimeric between Pan 6 and Pan 7 where (a) the hexonprotein of Pan 7 was replaced by that of Pan 6 (termed C767), (b), thefiber protein of Pan7 was replaced by that of Pan 6 (termed C776), (c)both the hexon and fiber proteins of the Pan 7 vector have been replacedby those from Pan 6 (termed C766).

The chimeric virus termed C767 was constructed essentially as describedabove for the C5C1C5 virus in Example 1. However, due to substantialhomology between the Pan6 and Pan7 sequences 5′ to the hexon sequence,it was not necessary to substitute the 5′ end of the genome between thepenton and the pol gene.

The chimeric vector C767 was compared to the C776, C766, the parent C6,and the parent C7, each expressing GFP.

Balb/C mice (25 per group) were immunized intramuscularly with eitherPan 6 or Pan 7 (10¹⁰ particles). Re-administration (10¹¹ particles i.v.,by tail vein injection) was attempted 3 weeks later using each of thefive GFP expressing vectors (C6-GFP, C7-GFP, and the three chimericvectors). Three days later the level of liver transduction was estimatedqualitatively by examining liver sections for the presence of GFPexpression and quantitatively by estimating copies of GFP DNA by Taqmananalysis. Administration of either one of the two chimpanzee adenovirusvectors does not affect the transduction efficiency of the other vector,while re-administration of the same vector is severly compromised. Thedata showed that antibodies to both hexon and fiber are important inpreventing re-administration of adenoviral vectors.

All publications cited in this specification are incorporated herein byreference, as are the priority documents, U.S. Patent Application60/575,429, filed Mar. 28, 2004; U.S. Patent Application No. 60/566,212,filed Apr. 28, 2004, and U.S. patent application Ser. No. 10/465,302,filed Jun. 20, 2003. While the invention has been described withreference to a particularly preferred embodiment, it will be appreciatedthat modifications can be made without departing from the spirit of theinvention. Such modifications are intended to fall within the scope ofthe appended claims.

1. A method of efficiently culturing a chimeric adenovirus in a selectedhost cell, said chimeric adenovirus being from a parental adenovirusstrain incapable of efficient growth in said host cell, said methodcomprising the steps of: (a) generating a chimeric adenoviruscomprising: (i) adenovirus sequences of the left terminal end and rightterminal end of a first adenovirus which grows in a selected host celltype, said left end region comprising the 5′ inverted terminal repeat(ITRs), and said right end region comprising the 3′ inverted terminalrepeat (ITRs); and (ii) the internal regions from a parental adenoviruswhich lacks its native 5′ and 3′ terminal regions, said internal regionscomprising the late genes encoding the penton, hexon, and fiber; whereinthe resulting chimeric adenovirus comprises, from 5′ to 3′, a leftterminal region of the first adenovirus, the internal region of theparental adenovirus, and the right terminal region of the firstadenovirus; and b) culturing said chimeric adenovirus in the presence offunctional adenovirus E1a, E1b, and E4 ORF6 genes from the firstadenovirus or from an adenovirus serotype which transcomplements thefirst adenovirus, and further in the presence of necessary adenoviralstructural genes from the left end of the adenovirus.
 2. The methodaccording to claim 1, wherein the internal region of the parentaladenovirus further comprises one or more functional adenovirus genesselected from the group consisting of Endoprotease open reading frame,DNA binding protein, 100 kDa scaffolding protein, 33 kDa protein,protein VIII, pTP, 52/55 kDa protein, protein VII, Mu and protein VI. 3.The method according to claim 1, wherein the polymerase, terminalprotein and 52/55 kDa protein functions are provided in trans.
 4. Themethod according to claim 1, wherein the first adenovirus furthercomprises the polymerase, terminal protein and 52/55 kDa proteinfunctions.
 5. The method according to claim 1, wherein the chimericadenovirus comprises the adenoviral late genes 1, 2, 3, 4, and 5 of theparental adenovirus.
 6. The method according to claim 1, wherein theselected host cell stably contains one or more of the adenovirus E1a,E1b or E4 ORF6 functions.
 7. The method according to claim 1, whereinthe chimeric adenovirus comprises one or more of the adenovirus E1a, E1bor E4 ORF6 of the first adenovirus.
 8. The method according to claim 1,wherein the first adenovirus is of human origin.
 9. The method accordingto claim 1, wherein the first adenovirus is of simian origin.
 10. Themethod according to claim 1, further comprising the step of isolatingthe chimeric adenovirus.
 11. A method for generating a chimericadenovirus for growth in a selected host cell, said chimeric adenovirusbeing derived from a parental adenovirus strain incapable of efficientgrowth in said host cell, said method comprising the step of generatinga chimeric adenovirus comprising: 5′ and 3′ terminal regions of a firstadenovirus which grows in a selected host cell type, said 5′ terminalregions comprising the 5′ inverted terminal repeat (ITRs) and necessaryE1 gene functions, and said 3′ terminal regions comprising invertedterminal repeat (ITRs) and necessary E4 gene functions; and internalregions from a parental adenovirus which lacks its native 5′ and 3′terminal regions, said internal regions comprising the hexon, pentonbase and fiber; wherein the resulting chimeric adenovirus comprises,from 5′ to 3′, the 5′ terminal region of the first adenovirus, theinternal region of the parental adenovirus, and the 3′ terminal regionsof the first adenovirus.
 12. A chimeric adenovirus produced according tothe method of claim
 1. 13. A chimeric adenovirus comprising a hexonprotein of a selected adenovirus serotype which is incapable ofefficient growth in a selected host cell, said modified adenoviruscomprising: (a) adenovirus sequences of the left terminal end of a firstadenovirus which grows in a selected host cell type, said left endregion comprising the E1a, E1b and 5′ inverted terminal repeat (ITRs);(b) adenovirus sequences of the internal region of the selectedadenovirus serotype which is incapable of efficient growth in theselected host cell, said internal region comprising the genes encodingthe penton, hexon and fiber of the selected adenovirus; (c) adenovirussequences of the right terminal end of the first adenovirus, said rightend region comprising the necessary E4 gene functions and the 3′inverted terminal repeat (ITRs), wherein the resulting chimericadenovirus comprises adenoviral structural and regulatory proteinsnecessary for infection and replication.
 14. The chimeric adenovirusaccording to claim 13, wherein the chimeric adenovirus further comprisesthe IIIa, 52/55 kDa and terminal protein (pTP) of the selectedadenovirus serotype.
 15. The chimeric adenovirus according to claim 13,wherein chimeric adenovirus comprises the polymerase of the firstadenovirus.
 16. The chimeric adenovirus according to claim 13, whereinthe chimeric adenovirus expresses a functional chimeric protein formedfrom the first adenovirus and the selected adenovirus, said chimericprotein is selected from the group consisting of polymerase, terminalprotein, 52/55 kDa protein, and IIIa.
 17. The chimeric adenovirusaccording to claim 13, wherein the chimeric adenovirus comprises theterminal protein, 52/55 kDa, and/or IIIa of the selected adenovirus. 18.A host cell comprising a chimeric adenovirus according to claim
 12. 19.The host cell according to claim 18, wherein said host cell is a humancell.
 20. An isolated simian adenovirus nucleic acid sequence selectedfrom the group consisting of: (a) SA18 having the sequence of nucleicacids 1 to 31967 of SEQ ID NO:12 and (b) a nucleic acid sequencecomplementary to the sequence of any of (a) to (f).
 21. An isolatedsimian adenovirus serotype nucleic acid sequence selected from one ormore of the group consisting of: (a) 5′ inverted terminal repeat (ITR)sequences; (b) the adenovirus E1a region, or a fragment thereof selectedfrom among the 13S, 12S and 9S regions; (c) the adenovirus E1b region,or a fragrnent thereof selected from among the group consisting of thesmall T, large T, IX, and IVa2 regions; (d) the E2b region; (e) the L1region, or a fragment thereof selected from among the group consistingof the 28.1 kD protein, polymerase, agnoprotein, 52/55 kD protein, andma protein; (f) the L2 region, or a fragment thereof selected from thegroup consisting of the penton, VII, VI, and Mu proteins; (g) the L3region, or a fragment thereof selected from the group consisting of theVI, hexon, or endoprotease; (h) the 2a protein; (i) the L4 region, or afragment thereof selected from the group consisting of the 100 kDprotein, the 33 kD homolog, and VIII; (j) the E3 region, or a fragmentthereof selected from the group consisting of E3 ORF1, E3 ORF2, E3 ORF3,E3 ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8, and E3 ORF9; (k) the L5region, or a fragment thereof selected from a fiber protein; (l) the E4region, or a fragment thereof selected from the group consisting of E4ORF7, E4 ORF6, E4 ORF4, E4 ORF3, E4 ORF2, and E4 ORF1; and (m) the 3′ITR, of any of SA18 SEQ ID NO: 12, or a sequence complementary to any of(a) to (m).
 22. A simian adenovirus protein encoded by the nucleic acidsequence according to claim
 21. 23. A composition comprising a simianadenovirus capsid protein according to claim 22 linked to a heterologousmolecule for delivery to a selected host cell.
 24. A method fortargeting a cell having an adenoviral receptor comprising delivering toa subject a composition according to claim
 23. 25. A nucleic acidmolecule comprising a heterologous simian adenoviral sequence accordingto claim
 21. 26. The nucleic acid molecule according to claim 25,wherein said simian adenoviral sequence encodes an adenoviral geneproduct and is operatively linked to regulatory control sequences whichdirect expression of the adenoviral gene product in a host cells. 27.The nucleic acid molecule according to claim 25, wherein said simianadenoviral sequence comprises the E1a region of SA18 SEQ ID NO:12.
 28. Apharmaceutical composition comprising the nucleic acid moleculeaccording to claim 27 and a physiologically compatible carrier.
 29. Arecombinant adenovirus having a capsid comprising a protein selectedfrom the group consisting of: (a) a hexon protein of SA18, SEQ ID NO 13,or a unique fragment thereof; (b) a penton protein of SA18, SEQ ID NO:14, or a unique fragment thereof; (c) a fiber protein of SA18, SEQ IDNO: 15, or a unique fragment thereof.
 30. The recombinant adenovirusaccording to claim 29, wherein the capsid is of an artificial serotype.31. The recombinant adenovirus according to claim 29, wherein said virusfurther comprises a heterologous gene operatively linked to sequenceswhich direct expression of said gene in a host cell.
 32. The recombinantadenovirus according to claim 29, further comprising 5′ and 3′adenovirus cis-elements necessary for replication and encapsidation. 33.The recombinant adenovirus according to claim 29, wherein said vectorlacks all or a part of the E1 gene.
 34. A host cell comprising aheterologous nucleic acid molecule comprising the nucleic acid sequenceaccording to claim
 21. 35. The host cell according to claim 34, whereinsaid host cell is stably transformed with the nucleic acid molecule. 36.The host cell according to claim 34, wherein said host cell expressesone or more adenoviral gene products from said nucleic acid molecule,said adenoviral gene products selected from the group consisting of E1a,E1b, E2a, and E4 ORF6.
 37. The host cell according to claim 34, whereinsaid host cell is stably transformed with a nucleic acid moleculecomprising the simian adenovirus inverted terminal repeats.
 38. Acomposition comprising a recombinant virus according to claim 29 in apharmaceutically acceptable carrier.
 39. A method for delivering aheterologous gene to a mammalian cell comprising introducing into saidcell an effective amount of the recombinant virus according to claim 29.40. A method for repeat administration of a heterologous gene to amammal comprising the steps of: (a) introducing into said mammal a firstvector which comprises the heterologous gene and (b) introducing intosaid mammal a second vector which comprises the heterologous gene;wherein at least the first virus or the second vector is a virusaccording to claim 29 and wherein the first and second recombinantvector are different.
 41. A method for producing a selected gene productcomprising infecting a mammalian cell with the recombinant virusaccording to claim 29, culturing said cell under suitable conditions andrecovering from said cell culture the expressed gene product.
 42. Amethod for eliciting an immune response in a mammalian host against aninfective agent comprising administering to said host an effectiveamount of the recombinant adenovirus of claim 29, wherein saidheterologous gene encodes an antigen of the infective agent.
 43. Themethod according to claim 42, comprising the step of priming the hostwith a DNA vaccine comprising the heterologous gene prior toadministering the recombinant adenovirus.